Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor.

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

Neonatal screening for galactosemia by quantitative analysis of hexose monophosphates using tandem mass spectrometry: a retrospective study.

BACKGROUND: Classic galactosemia (OMIM 230400) is an inherited disorder in the metabolism of galactose caused by deficiency of the enzyme galactose 1-phosphate uridyl transferase (EC 2.7.7.12). Galactosemia leads to accumulation of galactose and galactose 1-phosphate (gal-1-P) in blood and tissues and, if untreated, produces neonatal death or severe mental retardation, cirrhosis of the liver, and cataracts. Hence, the disorder is included in many neonatal screening programs. METHODS: We retrospectively analyzed filter-paper blood samples obtained 4-8 days postpartum for routine neonatal screening from 12 galactosemia patients and 2055 random controls. Total hexose monophosphates (HMPs) were used as a marker of gal-1-P and were assayed by negative-ion mode electrospray tandem mass spectrometry (tandem MS) with settings biased toward gal-1-P detection. The predominant precursor/product ion pair m/z 259/79 was used to quantify total HMPs by external standardization. RESULTS: Linear calibration curves were obtained in the range 0-8 mmol/L gal-1-P. The detection limit was 0.1 mmol/L HMP, and total CVs ranged from 13% at the detection limit to <8% at >1 mmol/L HMP. The method was in agreement with an alkaline phosphatase-galactose dehydrogenase method. All samples from galactosemia patients contained increased HMP concentrations (range for patients, 2.6-5.2 mmol/L; range for reference group, <0.10-0.94 mmol/L). The diagnostic sensitivity and specificity were 100% at a cutoff of 1.2 mmol/L HMP. A Duarte/classic galactosemia compound heterozygous sample could be discriminated clearly from both patient and reference samples. CONCLUSION: Quantitative analysis of HMPs by tandem MS can be used in laboratory investigations of galactosemia.

Effects of an aldose reductase inhibitor on erythrocyte fructose 3-phosphate and sorbitol 3-phosphate levels in diabetic patients.

Fructose 3-phosphate and sorbitol 3-phosphate are novel metabolites that have been shown to associate with the polyol pathway in animal experiments. Fructose 3-phosphate is of particular interest because of its potent glycation capability as compared with other glycolytic intermediates, e.g., fructose. We observed the effects of treatment with epalrestat, an aldose reductase inhibitor, on their concentrations in erythrocytes from diabetic patients. The levels of both metabolites were significantly higher in diabetic patients than in non-diabetic subjects. A group of patients who had been treated with epalrestat showed significantly lower levels of both metabolites as compared with those untreated. A treatment of three patients with epalrestat for one month resulted in obvious decreases in their concentrations. The results suggest a possible explanation for the preventive effect of an aldose reductase inhibitor on nonenzymatic glycation.

The measurement of enzyme activities in the resting human polymorphonuclear leukocyte--critical estimate of a method.

As a system for study, the isolated human polymorphonuclear leukocyte combines the advantages of a quasi-non-invasive preparation with a nearly complete complement of enzymes of carbohydrate and energy metabolism. However, small sample volumes and, in some cases, very low enzyme activities make high demands on sample processing, storage, and performance of continuous measurements, if the enzyme activities are to be measured with acceptable reproducibility. In the presented study several aspects of homogenization, storage, and continuous measurement were scrutinized, to identify critical steps and consider ways of optimizing the method. Polymorphonuclear leukocytes were separated from the blood of healthy subjects by sedimentation and density gradient centrifugation. After ultrasonic homogenization, 13 enzymes of glycolysis and gluconeogenesis, the tricarboxylic acid cycle, and glycogen metabolism were determined photometrically. The variation of several conditions showed: 1. The duration of exposure to ultrasound for the homogenization of polymorphonuclear leukocytes has no influence over a wide range of time. 2. Addition of the detergents Triton X-100 and deoxycholic acid, as well as the SH-group protector dithiothreitol, to the homogenizing medium increased the measured activities of only a few enzymes. 3. Considerable inaccuracy was encountered when the suspension was divided into parts for homogenization with different additives; such splitting of the suspension should therefore be performed only when necessary, as in the determination of reference values (e.g. protein or DNA content of the cell suspension). 4. Twenty four-fold determination of enzyme activities from one homogenate resulted in precisions between 4.5% (citrate synthase) and 14.4% (transketolase), which is satisfactory for the low activities (as low as 1 U/l) in the homogenate. 5. The reproducibility of enzyme activities, measured in homogenates of polymorphonuclear leukocytes from different blood samples drawn simultaneously, was only slightly worse than that of the continuous measurement method itself. Thus, the precision of the measurement of enzyme activity seems to be the main determinant of the overall method. In conclusion, the described procedure of separation, homogenization, and enzyme measurement in human polymorphonuclear leukocyte meets the requirements of biochemical or clinical trials and can be recommended for clinical metabolic studies.

Construction and characterization of adriamycin-loaded canine red blood cells as a potential slow delivery system.

Adriamycin was internalized in canine red blood cells (RBC) by two procedures involving (a) simple diffusion of the drug into cells and (b) hypotonic dialysis followed by isotonic resealing. The two procedures yielded comparable amounts of encapsulated adriamycin, around 35 micrograms/10(9) RBC. Exposure of adriamycin-loaded RBC to 0.16% glutaraldehyde consistently slowed down the rate of efflux of the drug as compared with non-glutaraldehyde-treated cells: after 1 h of incubation at 37 degrees C, greater than 80% of adriamycin was still present inside the glutaraldehyde-treated RBC, while at 24 h it was 66%, compared to 10% and 1%, respectively, in the adriamycin-loaded, non-glutaraldehyde-treated cells. Canine RBC showed a higher rate of transformation of adriamycin than the human cells, the only intracellular metabolite being adriamycinol, which is apparently formed by the NADPH-dependent enzyme aldehyde reductase. Production of adriamycinol was remarkably lower in the glutaraldehyde-treated RBC, as a result of progressive and extensive inactivation of hexose monophosphate shunt activity responsible for NADPH formation. These results, coupled with the known selective targeting of glutaraldehyde-treated RBC to liver, hold promise as to in vivo applications of this drug delivery system in antineoplastic therapy.

Identification of sorbitol 3-phosphate and fructose 3-phosphate in normal and diabetic human erythrocytes.

Using 31P NMR spectroscopy, we have identified sorbitol 3-phosphate and fructose 3-phosphate in normal human erythrocytes wherein their concentrations are estimated to be 13 mumol/liter cells. Incubation of hemolysates with sorbitol, fructose and ATP suggest that both sorbitol and fructose are phosphorylated separately and directly at the 3-hydroxyl position suggesting the presence in these cells of a novel and specific kinase(s). In addition to sorbitol 3-phosphate and fructose 3-phosphate which were previously identified in the mammalian lens and sciatic nerve, erythrocytes have two extra metabolites resonating at 6.7 and 6.8 ppm in the 31P NMR spectrum. Although not identified in this study, the unusual chemical shifts of these compounds, their low pKa values and the fact that they appear as doublet in proton-coupled 31P NMR spectra, suggest that these phosphomonoesters belong to the same class of metabolites as sorbitol 3-phosphate and fructose 3-phosphate. Preliminary studies of erythrocytes from an unselected group of diabetic subjects showed an overall increase in the concentration of all four metabolites, although an overlap with normal values was noted.

The insulin-like growth factor II/mannose-6-phosphate receptor is present in fetal and maternal sheep serum.

A large mol wt binding protein for insulin-like growth factor II (IGF-II) has been described in fetal sheep serum. We now provide evidence to demonstrate that this binding protein is the IGF-II/mannose-6-phosphate (Man-6-P) receptor. Serum and plasma were gel filtered on Sephadex G-200, and the column fractions were assayed for binding of radiolabeled IGF-II. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 150K and 40K carrier proteins. Binding to the void volume fractions was increased in fetal serum as well as maternal serum and dramatically decreased in the nonpregnant adult. Competitive binding studies with [125I]IGF-II and the void volume pools from fetal and maternal sheep serum demonstrated that IGF-I competed less potently than IGF-II, and insulin did not compete. There was no specific binding of [125I]IGF-I to the void volume pools of either fetal or maternal samples. Chemical cross-linking of [125I]IGF-II to aliquots of the void volume pools from fetal and maternal sheep serum samples and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol demonstrated a specific band at about 240K. Western blotting using a specific antiserum (no. 3637) against rat IGF-II/Man-6-P receptor was performed on aliquots of the Sephadex G-200 void volume pools of fetal, maternal, uterine vein, and adult sheep serum; a band of approximately 210K (without dithiothreitol) was seen. The IGF-II/Man-6-P receptor band was more intense in fetal serum than in either maternal or adult nonpregnant sheep serum. There was also increased binding of [125I]IGF-II in the 40K region of the Sephadex G-200 column fractions in the maternal serum compared to that in serum from nonpregnant adult ewes. When fetal, maternal, and adult nonpregnant sheep serum Sephadex G-200 pools were gel filtered on Sephadex G-50 in 1 mol/liter acetic acid to separate bound from free IGF, and IGF-II was measured by RRA, approximately 50% of the circulating IGF-II was associated with this IGF-II/Man-6-P receptor in fetal serum compared to 7% in maternal serum and 3% in adult nonpregnant serum. These data demonstrate that the IGF-II/Man-6-P receptor circulates in fetal sheep serum as well as maternal serum and appears to be a significant carrier for IGF-II in fetal sheep.

Improved metabolic properties of hexokinase-overloaded human erythrocytes.

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Oral intake of glucose plus galactose and erythrocyte galactose-1-phosphate. A nutritional evaluation of hydrolyzed lactose.

This study deals with the metabolic effects of hydrolyzed lactose: After an overnight fast 5 healthy adult volunteers consumed a glucose-galactose mixture equivalent to 61.4 g of lactose (or 125 g of a dried skim milk powder with hydrolyzed lactose). The postprandial rise of erythrocyte galactose-1-phosphate (gal-1-P) never exceeded 22.3 mumol per liter packed red blood cells. This amounts to no more than 22% of the levels known from galactosemic children to be safe, concerning ocular, neural or hepatic damage. We conclude that the consumption of the hydrolyzed lactose does not cause a risk for consumer's health as judged from this galactose metabolite. A considerably higher risk, however, may accompany the consumption of galactose alone which causes around 17-fold higher plasma galactose levels and around 8-fold higher erythrocyte gal-1-P concentrations for more extended time periods.

Microassay for estimation of galactose and galactose-1-phosphate in dried blood specimens.

A fluorometric assay for blood galactose and galactose-1-phosphate has been modified and improved to shorten the analysis time and to increase sensitivity above other published methods. The method may be useful as a quantitative screening or routine clinical test to detect infants suspected of having a defect of galactose metabolism. It can also be used to monitor blood galactose or galactose-1-phosphate levels in children with galactosemia who are on a lactose-free diet.

A modified method for the estimation of galactose-1-phosphate.

The measurement of galactose-1-phosphate has been advocated for monitoring galactosaemia. A radioenzymatic method is described in which red-cell haemolysates are incubated with radiolabelled uridine diphospho-glucose and exogenous galactose-1-phosphate uridyl transferase. Labelled glucose-1-phosphate is formed in proportion to the amount of galactose-1-phosphate present and is separated from the labelled uridine diphospho-glucose by affinity chromatography. The method is reproducible and has a low limit of detection. This allows it to be carried out on 100 microL of packed red cells.

Inhibition of the respiratory burst of human neutrophils by the polyamine oxidase-polyamine system.

The addition of the polyamines, spermine and spermidine, to human neutrophils caused a depression of the hexose-monophosphate (HMP) shunt activity of neutrophils stimulated with latex particles but not of unstimulated cells. The effect was dependent on the presence of bovine serum and was not observed when normal human serum was substituted for bovine serum. The polyamine oxidase (PAO) in bovine serum was probably responsible for generating the activity since normal human serum lacks PAO. A role for PAO was further supported by the finding that partially purified bovine PAO in the presence of polyamines similarly mediated inhibition of HMP shunt activity in stimulated neutrophils. Catalase failed to prevent the inhibitory effects of the PAO-polyamine system suggesting that H2O2 is not the responsible product. In addition, our results show that human pregnancy serum known to contain PAO activity in the presence of polyamines mediated a similar inhibition of the respiratory burst.

Limiting role of 6-phosphogluconolactonase in erythrocyte hexose monophosphate pathway metabolism.

The natural product of the glucose-6-phosphate dehydrogenase reaction is 6-phosphoglucono-delta-lactone, which must be hydrolyzed to 6-phosphogluconic acid before it can be further metabolized by 6-phosphogluconate dehydrogenase. Because this lactone is very unstable, it has been uncertain whether the enzyme that hydrolyzes it, 6-phosphogluconolactonase, is required for functioning of the hexose monophosphate pathway. We have purified glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase from human erythrocytes to the point where each enzyme is essentially free of each of the other activities. We constructed an artificial hexose monophosphate pathway from these enzymes, providing as substrate 14C-labeled glucose-6-phosphate either directly or by continual generation from 14C-glucose by yeast hexokinase and adenosine triphosphate. The oxidation of 6-phosphogluconic acid was estimated by measuring the CO2 formed. In the absence of a reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidizing system, such as oxidized glutathione (GSSG)-glutathione reductase or phenazine methosulfate, little CO2 was formed, and the presence of 6-phosphogluconolactonase did not affect the amount that was produced. When the hexose monophosphate pathway was stimulated by providing an NADPH-oxidizing system, CO2 was produced two and a half to five times as fast in the presence of 6-phosphogluconolactonase as in its absence. These studies suggest that 6-phosphogluconolactonase is required for the functioning of the hexose monophosphate pathway when the rate of oxidation of NADPH is accelerated.

The effects of hypothermia on neutrophil function in vitro.

Hypothermia may be associated with compromised host defenses and serious bacterial infections in man. We have examined the effects of moderate hypothermia (29 degrees C) on neutrophil function in vitro. At 29 degrees C, neutrophil phagocytosis of Staphylococcus aureus was impaired. In contrast, neutrophil killing of Streptococcus faecalis was most affected by hypothermia. Phagocytosis, as measured by neutrophil ingestion of opsonized oil-red-O-particles, was reduced at 29 degrees C over the 15 min of observation. Neutrophil metabolism linked to bactericidal pathways dependent on oxidative metabolism was reduced at 29 degrees C. Hexose monophosphate pathway (HMP) activity in neutrophils early after stimulation with latex particles was reduced. After 2 hr HMP activity was similar at 29 degrees C and 37 degrees C. Neotetrazolium dye reduction was reduced early after latex stimulation of neutrophils and after 30 min it was similar to cells at 37 degrees C. Leukocyte migration under agarose to bacterial-derived and formyl-methionyl-phenylalanine chemotactic factors was reduced by 50% and 70%, respectively. Migration to serum-derived chemotactic factor was reduced by only 20%. When cells were cooled to 29 degrees C for 30 to 90 min and rewarmed, neutrophil function was normal. These effects of hypothermia on neutrophil function may explain, in part, the increased incidence of serious and frequently fatal bacterial infections in man.

Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.

Effect of Coxiella burnetii on the stimulation of hexose monophosphate shunt and on superoxide anion production in human polymorphonuclear leukocytes.

Killed Coxiella burnetii (C.b.) cells in phase II but not in phase I had a mild stimulatory effect on hexose monophosphate shunt (HMPS) and superoxide anion production by human polymorphonuclear (PMN) leukocytes. Preincubation of C.b. cells of either phase with serum of a leukocyte donor lacking detectable antibodies to C.b. did not affect the studied activities of PMN leukocytes. In contrast, both HMPS stimulation and superoxide production were enhanced by specific opsonization of C.b. cells with rabbit immune sera containing corresponding phase I and/or phase II antibodies. Stimulatory effect was observed also with lipopolysaccharide-protein-phospholipid (LPS-Pr-Pl) complex but not with lipopolysaccharide-protein (LPS-Pr) complex and with purified lipopolysaccharide (LPS) isolated from phase I C.b. cells. Possible consequences of these findings for explanation of C.b. resistance to intracellular killing by professional phagocytes are discussed.

Glucose-6-phosphate-dehydrogenase-deficient erythrocytes have an impaired shape recovery mechanism.

In the human erythrocyte, the maintenance of the biconcave disc shape is important for cell viability as well as cell function. Previous studies have indicated the involvement of the hexose monophosphate shunt in the recovery of discoid shape after perturbation of echinocytic agents. In glucose-6-phosphate-dehydrogenase-deficient (Gd-) erythrocytes, the shunt activity is significantly decreased; thus, it might be expected that the shape recovery rate of Gd- erythrocytes would be decreased. We show here that shape recovery rates in the presence of the shunt stimulator methylene blue are as much as fivefold lower in Gd- erythrocytes. We also show that the protease inhibitor, N-alpha-tosyl-1-phenylalanine-chloromethyl ketone, has no effect on shape recovery in Gd-, whereas it increases normal cell shape recovery rates by 10-30-fold at 50 microM and causes cupping at 200 microM (see companion article by Alhanaty et al.). These changes are not due to reticulocytosis, as other hemolytic disorders do not show such changes. Further, both chronic hemolyzing Gd and A Gd variants show similar abnormal shape recovery behavior, whereas the extent of hemolysis is quite different between variants. Thus, the activity of the hexose monophosphate shunt appears to have a dramatic effect on the rate of reversal of echinocytosis. The lack of shunt activity of Gd cells would necessarily impair their ability to recover normal shape after perturbation.

Enhancement of neutrophil response by SH-containing compounds: modulation of superoxide and hydrogen peroxide production.

Anti-inflammatory effects of SH compounds in vivo and their effects on lymphocytes and macrophages in vitro have been described, but little is known about the mechanism of action or their effects on the neutrophil. In the present study the activity of seven low molecular weight non-protein SH compounds was compared. At a concentration of 3 X 10(-4)M all the compounds enhanced the activity of the HMP shunt of zymosan-stimulated neutrophils by 26-48% and that of PMA-stimulated cells by 6-44% above the control value (14.2 nmol CO2/2.5 X 10(6) neutrophils/30 min). Pretreatment of neutrophils with SH compounds for 15 min resulted in enhanced release of O-.2 by stimulated neutrophils in all cases, with the exception of GSH, by up to 87% above that of control. These effects were largely related to the ability of the compounds to modulate the release of O-.2 and H2O2 by stimulated neutrophils when present in the reaction mixture. Only the compounds alpha-MPG and cysteine had a mild preserving effect on the intracellular GSH concentration of stimulated neutrophils. None of the compounds tested had any adverse effect on phagocytosis or killing of opsonized bacteria by the neutrophils. SH compounds may protect sensitive SH groups of functional proteins by providing an easily accessible source of oxidizable SH groups in times of high oxidative stress, and their ability to interact with oxygen products could in part explain their anti-inflammatory properties.

Human polymorphonuclear leukocyte interaction with cyclosporine A.

The effects of cyclosporin A (cyA) on human polymorphonuclear leukocyte function, including phagocytosis, its associated metabolic burst, bacterial killing, and chemotaxis, were evaluated. Both Pseudomonas aeruginosa and Staphylococcus aureus were used as test particles. Polymorphonuclear leukocytes incubated in 10 and 50 micrograms of cyA per ml behaved normally with respect to phagocytosis and hexose monophosphate shunt activity at both high (10:1) and low (2:1) S. aureus/leukocyte ratios. With a small bacterial inoculum, killing of S. aureus was slightly impaired at early times only in the presence of 50 micrograms of cyA per ml. Phagocytosis and killing of P. aeruginosa with both large and small bacterial inocula were unaffected by cyA. Chemotaxis was within normal limits under all conditions. In addition, polymorphonuclear leukocytes from four renal transplant recipients receiving both cyA and prednisone demonstrated normal metabolic bursts and bacterial killing with both small and large inocula of S. aureus.