Bioelectrical impedance analysis and diabetes mellitus: which correlation among fructosamine, glycosylated haemoglobin and exchangeable potassium.

AIM: Bioelectrical impedance analysis (BIA) can monitor diabetics suffering from the frequently occurring state of hyperglycemia, as this can cause alterations in the water distribution in the body. The aim of the present study was to evaluate the relationship between the composition of the body and the diabetic disease during decompensation through the impedanciometric analysis in diabetic patients of type 1 and type 2 and to understand the possible alterations of water distribution. METHODS: The study was carried out with 52 subjects (8 males, 44 females), average years 46.5; 15 of them were diabetic, 7 characterised by diabetes of type 1 and 8 by diabetes of type 2. All the patients recruited were in poor metabolic control (glycosylated hemoglobin [HbAlc] >6%). In order to avoid any errors during the evaluation ofa water distribution in the body, patients suffering from hypertension were excluded from the recruitment process. All patients underwent impedanciometry total body using the HUMAN IM SCAN apparatus multifrequency. RESULTS: Through the application of BIA, our work has shown how diabetic patients have a lower quantity of extracellular water (ECW) and exchangeable potassium (Ke) in the body, as compared to non-diabetic patients. The causes of this could be the alteration of the plasmic osmolarity and the possible reduction of the mass of metabolically active cells. Further-more a relationship between fructosamine in the blood and Ke has been found and, alongside another, more significant, between HbA1c and Ke. According to the opinions of the authors, such results are worthy for further studies in order to obtain a greater accuracy in the evaluation of the amount of Ke and an alternative in estimation of metabolic control.

Smoking is associated with reduced serum levels of the antioxidant enzyme, paraoxonase, in Type 2 diabetic patients.

AIMS: To analyse the association of smoking with paraoxonase (PON1) in Type 2 diabetic patients. METHODS: Type 2 diabetic patients were recruited independently in two centres (Ancona, Italy and Geneva, Switzerland) and serum PON1 mass and activities were assayed. Current smoking status was established and its association with serum PON1 analysed. RESULTS: Type 2 diabetic patients who smoked had significantly lower serum PON1 mass and activity. This was evident in both groups of patients, even though Swiss patients were composed of coronary patients. Multivariate analyses established that smoking was an independent determinant of serum PON1 status. CONCLUSIONS: Smoking is associated with reduced serum levels of the antioxidant enzyme, PON1, even against an already unfavourable background of diabetes and coronary disease. It suggests that a combination of smoking and diabetes may be particularly deleterious for PON1 and consequently for the anti-oxidant capacity of high-density lipoproteins.

Diabetes and non-traumatic lower extremity amputation in a region of central Italy.

As stated in the St. Vincent Declaration, the reduction of the number of lower extremity amputations (LEA) is one of the major targets in diabetes care. Little information on the prevalence of this complication is available in Italy at present. The aim of this study was to evaluate the impact of diabetes on LEA in a region of central Italy. The regional database of hospital discharge records was used to identify all the cases of non-traumatic LEA (Nt-LEA) that had occurred in 1997 and 1998. Diagnosis of diabetes and the type of surgical intervention were defined on the basis of the medical records. Amputations were categorised as minor or major according to the level of the surgical procedure. The duration of hospitalisation was used to analyse the impact of diabetes on the health care system. Prevalence of diabetes in people who underwent Nt-LEA was 55.9% and it was similar in patients who underwent minor or major amputation (58.7 and 55.0%, respectively). No difference between diabetic and non-diabetic patients was observed as regards sex, level of amputation and duration of hospitalisation. Age was the only predictor of diabetes in amputees. The lack of specific protocols for diabetes foot care or a non-homogeneous application of these protocols in the health care service could account for a similar prevalence of diabetes among patients who underwent minor or major amputation.

Apolipoprotein E polymorphisms and mortality in Italian type 2 diabetic patients.

AIMS: To determine if apolipoprotein E polymorphism is associated with cardiovascular or all-cause mortality in Italian Type 2 diabetic patients. METHODS: A prospective study of mortality in Type 2 diabetic patients (n = 433) as a function of apolipoprotein E phenotype, which was assessed at entry into the study. During follow up (10 years), 110 (25.4%) patients died of which 66 (15.2%) were the result of cardiovascular causes. Cause of death was established from death certificates and clinical records. The clinical status of the survivors was determined at the end of the study. RESULTS: Apolipoprotein E polymorphisms were not associated with excess cardiovascular or all-cause mortality in the Italian Type 2 diabetic patients either in univariate or multivariate analyses. Age, duration of diabetes and glycated haemoglobin levels at entry were the primary determinants of premature mortality in the diabetic population. CONCLUSIONS: Apolipoprotein E polymorphisms are not markers for premature mortality in Italian Type 2 diabetic patients. The impact of apolipoprotein E mutations may be attenuated by environmental factors, notably a healthier diet, in Italian patients.

Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects.

AIMS/HYPOTHESIS: The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. METHODS: The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 micromol/l homocysteine. Stimulation experiments were done in vitro by the addition of alpha-thrombin (0.2 U/ml). RESULTS: Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. CONCLUSION/INTERPRETATION: Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation.

Diagnosis of MODY in the offspring of parents with insulin-dependent and non-insulin-dependent diabetes mellitus.

Maturity Onset Diabetes of the Young (MODY) is an autosomal dominant monogenic form of type 2 diabetes mellitus (DM) representing 5% of youth-onset DM in the Caucasian population. In young adults the disease can be present as either non-insulin dependent or insulin-dependent DM. The diagnosis of this genetic disorder in children and adolescents is rare because of the mild glucose metabolism disorder at this time. We performed a metabolic, autoimmune and genetic study in 40 offspring of young parents affected by insulin-dependent DM (Group A) and in 35 offspring of young parents affected by early-onset non-insulin-dependent DM (Group B). Two children of Group A (5%) were found to be affected by fasting hyperglycemia and carry a GCK gene mutation that in one case was present also in the diabetic father. Eighteen offspring of Group B (51%) were positive for GCK or HNF-1alpha gene mutations present in the affected parents. All but two of these young patients had impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). Eleven of them were younger than 16 years. We conclude that screening for DM in youth should be extended to MODY in young families with both non-insulin-dependent and insulin-dependent DM. The sensitivity of the metabolic tests will precede the genetic diagnosis.

One- and two-compartment minimal models detect similar alterations of glucose metabolism indexes in hypertension.

A standard intravenous glucose tolerance test (IVGTT) was performed in 10 nondiabetic patients with essential hypertension (H group) and 9 normotensive control subjects (N group). A 2-compartment minimal model (2CMM) of glucose kinetics was applied to estimate indexes of glucose effectiveness, S2G and insulin sensitivity, S2I, by means of a maximum a posteriori (MAP) bayesian estimation technique. These estimates were contrasted to the S1G and S1I indexes provided by the classic minimal model (1CMM). In both the N group and the H group, the 2CMM underestimated the glucose effectiveness and overestimated the insulin sensitivity. In the H group, S2G was, on average, 63% of S1G (P > .05) and S2I was 137% of S1I (P > .05). In the N group S2G was 67% of S1G (P > .05) and S2I was 134% of S1I (P > .05). The 2CMM detected a reduction of approximately 40% (P > .05) and approximately 48% (P > .05) in S2G and S2I estimates, respectively, from the N group to the H group. Despite its reduced complexity, the 1CMM also detected a reduction of approximately 35% (P < .05) and approximately 49% (P < .05) in the S1G and in S1I indexes, respectively. Thus, the 1CMM and 2CMM showed a substantial equivalence in detecting a severe reduction in insulin sensitivity and impaired glucose effectiveness in hypertensive patients compared with normal.

Influence of body mass on glycemic control in a type 2 diabetic population: a 3-year follow-up.

Obesity is often associated with type 2 (non insulin-dependent) diabetes. A growing body of evidence support the hypothesis that these two diseases share a common pathogenesis. Nevertheless, experience derived from clinical observation on type 2 diabetic patients indicates that reduction of body weight is not always accompanied by an improvement in metabolic control and that a good metabolic control is often obtained without influencing body composition. Aim of the present study was to evaluate the relationship between body mass and glycemic control in a type 2 diabetic population by a 3 years observational study. A cohort of 562 subjects was studied. At entry more than 80% of patients were overweight or obese according to the body mass index (BMI) scale and this proportion was not significantly reduced at the end of the follow-up. At entry all patients had a glycosylated hemoglobin (HbA1c) value above 8.1% whereas at the end of follow-up more than 2/3 of patients were in good metabolic control. No relationship was observed between modification of body mass and metabolic control. These data confirm the high frequency of obesity among type 2 diabetic individuals but they suggest that impaired glucose metabolism and alteration of body weight have different pathogenesis.

Renal disease as a determinant of increased lipoprotein(a) concentrations in diabetic patients.

OBJECTIVE: This study examined the hypothesis that kidney function is an independent determinant of lipoprotein(a) [Lp(a)] concentrations in people with diabetes. RESEARCH DESIGN AND METHODS: Lp(a) concentrations were measured in plasma samples from 273 type 2 and 223 type 1 diabetic patients recruited from a diabetes clinic. Kidney function was categorized as normal or pathological according to plasma creatinine levels and creatinine clearance rates. RESULTS: Macroalbuminuria was uniformly associated with significantly raised plasma concentrations of Lp(a) regardless of the marker used to identify kidney dysfunction. In contrast, in patients with microalbuminuria, significantly raised plasma Lp(a) levels were observed only when creatinine clearance rates or plasma creatinine levels indicated pathological kidney function. These conclusions were independent of diabetes type. CONCLUSIONS: In microalbuminuria and apparently in normoalbuminuria, altered kidney function determined by creatinine clearance rates or creatinine levels appears to be a major determinant of raised Lp(a) levels in both type 1 and type 2 diabetic patients. In contrast, Lp(a) concentrations were uniformly raised in patients with macroalbuminuria.

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.

AIMS/HYPOTHESIS: The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. METHODS: Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting. RESULTS: In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. CONCLUSION/INTERPRETATION: Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.

Coronary heart disease, type 2 diabetes mellitus and cardiovascular disease risk factors: a study on a middle-aged and elderly population.

The risk for all the manifestations of atherosclerotic disease is increased in patients affected by type 2 diabetes mellitus. The aim of the present work was to evaluate the prevalence of coronary heart disease (CHD) in a well-characterized middle-aged and elderly Italian diabetic population. The population studied included 3862 subjects, i.e. all the patients affected by type 2 diabetes of age >or=50 years attending the outpatient diabetes care unit of INRCA in Ancona (Italy) from 1 August 1997 to 31 July 1998. We collected and analysed both clinical and laboratory data by means of a computerized data base for the outpatient clinic management. The prevalence rate of CHD was 20.25% in this population. The groups with CHD and without CHD did not differ significantly with respect to age at onset of diabetes, body mass index and HbA1c levels, while patients with CHD were significantly older than patients without CHD and had a longer duration of diabetes. The prevalence of patients with hypertension (52.9 vs 63.0%, P<0.001), hypercholesterolemia (11.6 vs 14.1%, P<0.05) and hyperlipidemia (17.8 vs 23.3%, P<0.001) was significantly higher in the group of diabetic subjects affected by CHD than in patients not affected by heart ischemic disease. It might be hypothesized that the improvement of metabolic profile and the currently feasible control of non-diabetic risk factors could reduce cardiovascular disease rates in type 2 diabetic patients.

Modifications induced by plasma from insulin-dependent diabetic patients and by lysophosphatidylcholine on human Na+,K(+)-adenosine triphosphatase.

To investigate the molecular mechanisms of the inhibition of Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) in diabetes mellitus, we incubated Na+,K(+)-ATPase purified from human placenta of six healthy nondiabetic women with plasma from six insulin-dependent diabetic (IDDM) men and six healthy controls and with different concentrations of lysophosphatidylcholine (LPC). We determined the enzyme activity, anthroyl ouabain-binding capacity, dissociation constant (Kd), and average lifetime values (tau) by the static and dynamic fluorescence of anthroyl ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylamino-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Moreover, we studied the lipid microenvironment surrounding the Na+,K(+)-ATPase purified from the placentas of six healthy women and six insulin-dependent diabetic women, determining the percent composition of phospholipids of the lipid annulus. The addition of total and protein-free IDDM plasma to normal Na+,K(+)-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1: 100), whereas the ouabain-binding capacity, Kd, and tau were not affected by IDDM plasma. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by diabetic plasma. The incubation of Na+,K(+)-ATPase with LPC caused an inhibition of the enzymatic activity without modifications of the anthroyl ouabain-binding capacity and dissociation constant. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by 5 mumol/L LPC. The study of the phospholipids surrounding Na+,K(+)-ATPase demonstrated a significant increase in the percent LPC content in IDDM patients compared with controls together with a concomitant decrease in phosphatidylcholine. These observations indicate that the inhibition caused by diabetic plasma on Na+,K(+)-ATPase is not dependent on a modification of the ouabain-binding site and that it seems to mimic the effect of LPC addition. A link between modification of the lipid moiety of the enzyme and Na+,K(+)-ATPase inhibition might be hypothesized.

Decreased nitric oxide synthase activity in platelets from IDDM and NIDDM patients.

Nitric oxide (NO) produced by platelet nitric oxide synthase (NOS) inhibits platelet activation by increased cytoplasmic cGMP levels. The aim of this study was to investigate platelet NOS activity in insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), which are characterized by enhanced platelet activation. HbA1c levels, platelet NOS and platelet membrane Na+/K+ ATPase activity were determined in 19 IDDM patients, 21 NIDDM patients and 31 healthy control subjects. NOS activity was measured by a spectrophotometric method based on NO-dependent oxidation of oxyhaemoglobin to met-haemoglobin. Na+/K+ ATPase activity was measured by the method of Kitao and Hattori. Both NOS and Na+/K+ ATPase activity were significantly reduced in diabetic subjects compared with control subjects. NOS showed a significant negative relation with HbA1c levels and a positive relation with Na+/K+ ATPase activity in diabetic patients. It is hypothesized that the decreased NOS activity might play a role in the pathogenesis of diabetic vascular complications.

Altered platelet membrane dynamic properties in type 1 diabetes.

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.

Modifications induced by insulin-dependent diabetes mellitus on human placental Na+/K+-adenosine triphosphatase.

The causes of the reduced activity of Na+/K+-adenosine triphosphatase (ATPase) in human diabetes are still the object of controversy. The aim of this work was to investigate the mechanisms of inhibition by means of the study of the Na+/K+-ATPase purified from human placenta. We purified Na+/K+-ATPase from term placentas of six healthy women and six age-matched women with insulin-dependent diabetes mellitus (IDDM) in good metabolic control. The enzymatic activity was reduced in both the microsomal fraction and the purified Na+/K+-ATPase obtained from diabetic women, whereas no difference was found in the number of active molecules determined by anthroyl ouabain binding. The Na+/K+-ATPase purified from women with IDDM did not show any modification in the ouabain affinity or changes in the physicochemical structure of the ouabain binding site investigated by dynamic fluorescence or alterations in lateral diffusion. The activation energy of the enzyme was increased, whereas the tryptophan accessibility of the enzyme was lower in women with IDDM. The fluidity of the lipid anulus of the enzyme was higher in women with IDDM than in control women, as suggested by fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene. The adenosine triphosphate-binding site, investigated by anisotropy decay studies of the fluorescent probe pyrene isothiocyanate, was modified in women with IDDM. It appears that the Na+/K+-ATPase of human placenta is altered in its disposition in IDDM.

Diabetes mellitus and subjects' ageing: a study on the ATP content and ATP-related enzyme activities in human erythrocytes.

Na+/K(+)- and Ca(2+)-ATPase are the major ATP-dependent membrane-bound enzymes that regulate the cation transmembrane gradient which is altered both in red blood cell (RBC) senescence and in RBCs of diabetic patients. In an attempt to clarify the possible connection between diabetes mellitus and ageing, we investigated the relationship between RBC ATP content, Na+/K(+)-ATPase, Ca(2+)-ATPase activities and ageing in healthy, insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) subjects. A significant correlation was found (r = -0.82; P < 0.001) between RBC ATP content and subject's age only in the control group. A significant reduction in Na+/K(+)-ATPase activity was observed in the older group (C2) of control subjects, in comparison with the younger (C1) one. In both IDDM and NIDDM subjects, the enzymatic activity was significantly decreased when compared with health subjects of similar age (P < 0.001). A significant negative correlation was found between age and enzymatic activity in healthy subjects (r = -0.60; P < 0.001). No difference was observed in the RBC membrane Ca(2+)-ATPase activity between younger (C1) and older (C2) healthy subjects. Ca(2+)-ATPase activity was significantly increased both in IDDM patients compared with C1 (P < 0.001) and in NIDDM patients compared with C2 (P < 0.001). The present data indicate that ageing causes a reduction in the erythrocyte ATP content in both healthy and diabetic subjects. In diabetic patients Na+/K(+)-ATPase activity decreases independently of age.

Diabetes mellitus and chronic heart failure.

Cardiovascular disease has a high prevalence in diabetic patients. Diabetes mellitus is an important risk factor for atherosclerosis and coronary disease mainly through obesity, hyperlipidemia, insulin-resistance, hyperinsulinemia, hyperglycemia and altered homeostasis. The correlation between diabetes and chronic heart failure is not widely documented in the literature. According to the Framingham study, the incidence of cardiovascular morbidity per year is 39.1% in diabetic males and 17.2% in diabetic females; chronic heart failure afflicts 7.6% of diabetic males and 11.4% of diabetic females. Actual knowledge about pathophysiology suggests that cardiac involvement in diabetes is not only related to macrovascular injury but also to other factors, such as alterations of autonomic nervous system, that can contribute to diabetic cardiopathy. The present study evaluated the prevalence of chronic heart failure in an Italian diabetic population in order to discuss the rationale of the therapeutic strategies.

Morphological alterations and increased resistance to hemolysis in t-butyl hydroperoxide incubated RBC from elderly subjects.

The response of human red blood cells (RBC) to oxidative stress has been studied with the aim to evaluate any difference in the behavior of cells from young and old subjects. Thus, RBC from 5 young (27 +/- 2 years) and 5 old (80 +/- 5 years) individuals have been treated with the organic peroxide t-butyl hydroperoxide (TBHP). The two groups behaved differently: after 4 hrs of incubation in 0.5 mM TBHP, RBC from young donors showed a higher level of hemolysis; instead, RBC from old individuals showed abnormal morphologies, being absent in unstressed RBC, with constriction and budding, which could be identified as poikilocytosis. The same abnormal forms are found in patients with spectrin mutation, leading us to hypothesize that TBHP causes damage to the cytoskeletal spectrin. This suggests that poikilocytosis might be an early stage of red blood cell hemolysis because their presence is associated to a lower level of hemolysis.