The squatting test. A useful tool to assess both parasympathetic and sympathetic involvement of the cardiovascular autonomic neuropathy in diabetes.

The heart rate responses observed after both squatting and standing are thought to be of reflex nature and may be useful to assess the functional integrity of parasympathetic and sympathetic nerves in diabetes. In the standard maneuver, each subject stood still for 3 min, then squatted down for 1 min, and at last stood up during an inspiratory phase. In 10 healthy subjects (25-31 years of age), lengthening of the R-R interval during squatting was abolished by atropine, whereas propranolol markedly attenuated shortening of the R-R interval at standing from squatting. Squatting test (SqT) ratios (SqT vagal [SqTv] = ratio between the R-R interval mean before squatting and the longest R-R interval after squatting; SqT sympathetic [SqTs] = ratio between the basal R-R interval and the shortest R-R interval at standing) were calculated in 558 healthy subjects and 346 diabetic patients (insulin-dependent diabetes mellitus/non-insulin-dependent diabetes mellitus: 103/243). Normal ranges (95 and 99% confidence intervals [CIs]) for subjects 20-74 years of age showed a statistically significant negative correlation with age. SqTv was outside the 99% CI in 145 (42%) diabetic patients and in 7 (1.3%) of the control subjects. The corresponding figures for SqTs were 40 and 0.8%, respectively. Age and duration of diabetes had a negative influence on SqT ratios. SqT ratios were compared with other reflex tests currently used for diagnosis of autonomic neuropathy: deep breathing (DB), lying-to-standing (LS), Valsalva manuever, and blood pressure change after standing (orthostatic hypotension [OH]).(ABSTRACT TRUNCATED AT 250 WORDS)

Tolrestat in the primary prevention of diabetic neuropathy.

OBJECTIVE: To compare the effects of tolrestat and placebo in patients with subclinical diabetic neuropathy. RESEARCH DESIGN AND METHODS: Non-insulin-dependent diabetes mellitus (NIDDM) patients with early involvement of the autonomic nervous system were identified by only one pathological (outside the 99% confidence interval of the normal population) squatting test (vagal or sympathetic). Fifty-seven patients entered a randomized, placebo-controlled, double-blind, parallel 52-week study of tolrestat at a dose of 200 mg/day. Cardiovascular reflex tests (squatting vagal and sympathetic tests, pressure gain, deep breathing, lying-to-standing, Valsalva maneuver, and orthostatic hypertension), vibration thresholds, tendon reflexes, and muscle strength were assessed throughout the study. RESULTS: At 12 months, nerve function significantly improved in patients receiving tolrestat and deteriorated in patients taking placebo. At baseline, the squatting vagal test was normal in 16 patients in the tolrestat group and in 15 patients in the placebo group. At 12 months, 25 patients taking tolrestat had a normalized squatting test, but only 6 patients taking placebo did (P = 0.02). Vibration perception threshold improved by a value of 6 +/- 3 V in the tolrestat group (P < 0.001) and deteriorated by a value of 3 +/- 1.8 V (P < 0.001) in the placebo group. CONCLUSIONS: Tolrestat may be useful in the primary prevention of diabetic neuropathy.

Detection of early sympathetic cardiovascular neuropathy by squatting test in NIDDM.

OBJECTIVE: To determine the role of the squatting test in the detection of early sympathetic neuropathy in patients with non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: Three groups of nonsmoking, nonobese subjects were studied: 10 healthy subjects, 10 NIDDM patients without autonomic neuropathy (AN), and 10 NIDDM patients with AN defined by the presence of a pathological deep-breathing value. All subjects were given three postural tests: lying-to-standing, sitting-to-standing, and squatting test. Heart rate (HR) and finger arterial pressure were recorded with a noninvasive technique. RESULTS: Blood pressure (BP) fall (expressed as decremental area) was not significantly different among the groups at standing up after sitting or lying. By contrast, a significantly greater BP drop occurred in NIDDM patients with AN (1,123 +/- 245 mm2) compared with NIDDM patients without AN (460 +/- 232 mm2) or normal subjects (429 +/- 138 mm2, P < 0.001). The HR increase after all the orthostatic maneuvers was smaller in diabetic patients with AN (P < 0.01) compared with that recorded in other groups. Significant correlations were observed between BP fall after squatting and either the expiration:inspiration ratio at deep breathing (r = -0.77, P < 0.001) or the duration of diabetes (r = 0.76, P < 0.001). CONCLUSIONS: The intrinsic orthostatic load of the squatting test, which is greater than conventional postural maneuvers, makes the squatting test an easy and useful test to detect early orthostatic dysregulation in NIDDM.

Metformin improves glucose, lipid metabolism, and reduces blood pressure in hypertensive, obese women.

OBJECTIVE: To determine the effects of metformin on blood pressure, left ventricular mass, and some metabolic and endocrine parameters in nondiabetic, obese, hypertensive women. RESEARCH DESIGN AND METHODS: Twelve obese, nondiabetic, hypertensive women received 850 mg metformin 2 times/day for 12 wk and placebo for another 12 wk, according to a double-blind, cross-over, randomized design. All patients were hospitalized 4 times, i.e., before randomization and after each treatment (metformin or placebo), to conduct metabolic and cardiovascular investigations (oral glucose tolerance test, euglycemic clamp associated with indirect calorimetry, and echocardiography). RESULTS: Fasting glucose, HbA1c, fasting and glucose-stimulated insulin, blood pressure and left ventricular mass, cholesterol, triglycerides, and fibrinogen decreased significantly after metformin treatment, whereas high-density lipoprotein cholesterol increased. The improvement in glucose metabolism resulted from increased sensitivity to insulin. CONCLUSIONS: These findings suggest that metformin treatment in obese, nondiabetic, hypertensive women produces a more favorable cardiovascular risk profile.

Low-dose iloprost infusion improves insulin action in aged healthy subjects and NIDDM patients.

OBJECTIVE: To investigate the effect of iloprost infusion on insulin action. RESEARCH DESIGN AND METHODS: Thirteen healthy subjects and 13 non-insulin-dependent diabetes mellitus (NIDDM) patients matched for age (68.2 +/- 0.5 vs. 67.9 +/- 0.5 years, NS), gender ratio (7 men:6 women vs. 6 men:7 women), body weight, body fat distribution, arterial blood pressure, and plasma triglyceride levels (1.89 +/- 0.09 vs. 1.87 +/- 0.08 mmol/l, NS) were studied. In eight healthy subjects and eight NIDDM patients, we studied insulin action by euglycemic glucose clamp (insulin infusion rate 2 mU.kg-1.min-1) along with saline and iloprost delivery (0.7 ng.kg-1.min-1). In the other five subjects of each group, forearm blood flow and insulin-mediated glucose uptake during saline and iloprost infusion (0.7 ng.kg-1.min-1) were investigated. RESULTS: Iloprost infusion improved insulin-stimulated whole-body glucose uptake and oxidative and nonoxidative glucose metabolism in both study groups. Forearm blood flow under basal conditions and with insulin infusion (2 mU.kg-1.min-1) did not show any significant difference from that during saline and iloprost infusion (0.7 ng.kg-1.min-1) in healthy subjects and diabetic patients. CONCLUSIONS: Iloprost infusion improves insulin action in healthy subjects and NIDDM patients.

Changes in glucose turnover parameters and improvement of glucose oxidation after 4-week magnesium administration in elderly noninsulin-dependent (type II) diabetic patients.

The aim of the present study was to investigate the effects of magnesium supplementation on glucose uptake and substrate oxidation in noninsulin-dependent (type II) diabetic patients. Nine elderly non-obese noninsulin-dependent (type II) diabetic patients, treated by diet only, participated in the study, which was designed as randomized, double blind, and cross-over. Each patient was followed up for a prestudy period of 3 weeks before inviting him/her to receive placebo or magnesium supplementation (15.8 mmol/day) for 4 weeks. At the end of each treatment period, a euglycemic hyperinsulinemic glucose clamp with simultaneous D-[3-3H]glucose infusion and indirect calorimetry was performed. Magnesium supplementation resulted in significantly increased plasma and erythrocyte magnesium levels, whereas body weight and fasting plasma glucose did not change. In the last 60 min of the glucose clamp, insulin-mediated glucose disappearance, total body glucose disposal (24.5 +/- 0.4 vs. 28.2 +/- 0.7 mumol/kg.min; P < 0.005), and glucose oxidation (13.0 +/- 0.4 vs. 16.3 +/- 0.8 mumol/kg.min; P < 0.01) were increased after chronic magnesium supplementation. Endogenous glucose production, nonoxidative glucose disposal, lipid and protein oxidation, and insulin MCR were not affected. In conclusion, a 4-week magnesium supplementation improves insulin sensitivity and glucose oxidation in the course of a euglycemic-hyperinsulinemic glucose clamp in noninsulin-dependent diabetic patients. Long term studies are needed to determine whether magnesium supplementation is useful in the management of type II diabetes.

Pharmacological doses of vitamin E and insulin action in elderly subjects.

Twenty elderly (77 +/- 0.4 y), nonobese [body mass index (in kg/m2) 26.4 +/- 0.5] subjects with normal glucose tolerance were submitted to a euglycemic hyperinsulinemic (3.5 pmol.min/kg) glucose clamp in a double-blind, crossover, randomized procedure after 4 mo treatment with either vitamin E (900 mg d-alpha-tocopherol/d, Ephynal; Roche, Milan, Italy) or placebo. Body mass index was practically unchanged throughout the study. After the glucose clamp, insulin-mediated stimulation 2 of whole-body glucose disposal (18.4 +/- 0.5 vs 26.1 +/- 0.6 mumol.min/kg lean body mass P < 0.02) was significantly potentiated by vitamin E rather than placebo administration. Furthermore, net changes in plasma vitamin E concentrations correlated with net changes in insulin-stimulated whole-body glucose disposal (r = 0.60 P < 0.003). Plasma vitamin E concentrations seem to play an important role in the modulation of insulin action in elderly people.

Chronic magnesium administration enhances oxidative glucose metabolism in thiazide treated hypertensive patients.

In newly-diagnosed untreated (n = 24) and thiazide treated (n = 18) hypertensive patients erythrocyte ion content and plasma ion and metabolite levels were determined. Thiazide treated patients had lower arterial blood pressure, plasma ion levels, erythrocyte magnesium and potassium content but higher fasting plasma insulin (66 +/- 7 v 87 +/- 8 pmol/L P < .02), triglycerides (1.88 +/- 0.24 v 2.34 +/- 0.44 mmol/L P < .05), free fatty acids (0.68 +/- 0.11 v 0.81 +/- 0.18 mmol/L P < .05). Subsequently, in a double-blind fashion and in random order thiazide diuretic treated patients were assigned to two groups: the first (n = 9) taking magnesium (15.8 mmol/day) and the other (n = 9) a placebo. Each treatment period lasted 8 weeks. At the end of each treatment period, each patient underwent blood sampling for determination of erythrocyte ion content and plasma ion and metabolite levels and was submitted to an euglycemic hyperinsulinemic (1 mU/kg/min for 120 min) glucose clamp. In this latter test D-3-H glucose infusion and indirect calorimetry allowed determination of glucose turnover parameters and substrate oxidation respectively. Chronic magnesium administration (CMA) raised fasting plasma (0.79 +/- 0.03 v 0.83 +/- 0.02 mmol/L, P < .05) and erythrocyte (1.98 +/- 0.08 v 2.35 +/- 0.03 mmol/L, P < .01) magnesium content. Along with insulin infusion, CMA improved glucose uptake, glucose metabolic clearance rate, and oxidative glucose metabolism. In the multiple linear regression analysis of the pooled basal data (n = 42), erythrocyte magnesium content displayed an independent correlation with basal plasma insulin levels (t = -2.08, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of nitrendipine and cilazapril in patients with hypertension and non-insulin-dependent diabetes mellitus.

The metabolic and cardiovascular effects of nitrendipine and cilazapril in patients who have elevated blood pressure and non-insulin-dependent diabetes mellitus (NIDDM) were compared. After at least 6 weeks of a washout period, 20 NIDDM patients who had diastolic blood pressure in the range of 90-105 mm Hg received a single-blind placebo for 4 weeks and then were randomized to receive 20 mg nitrendipine once daily and 5 mg cilazapril once daily each for 12 weeks according to a crossover, double-blind procedure. Nitrendipine and cilazapril reduced diastolic blood pressure levels 12% and 13%, left ventricular mass index (LVMI) levels 13% and 12%, and raised whole glucose disposal levels 18% and 19.5%, respectively. Only nitrendipine reduced glucose-stimulated insulin levels. Nitrendipine is as effective as cilazapril in lowering diastolic blood pressure and LVMI levels and in increasing glucose disposal levels in these patients.

Evidence of a cold immunoglobulin M autoantibody against 78-kD platelet glycoprotein in a case of EDTA-dependent pseudothrombocytopenia.

Pseudothrombocytopenia is a phenomenon in which the electronic count shows spuriously low platelet counts in subjects with normal platelet levels. The mechanism of anticoagulant-dependent pseudothrombocytopenia appears to involve cold reactive agglutinins against platelet antigens. The authors report a case of EDTA-dependent pseudothrombocytopenia with evidence of a cold immunoglobulin M antibody against 78-kD platelet membrane glycoprotein (GP). Cell counts were performed by Coulter Counter S-Plus VI (Coulter, Hialeah, FL) in the following anticoagulants: EDTA, Na-citrate, and citrate-theophylline-adenosine-dipyridamole. Anti-platelet antibodies and platelet membrane GP antigens were assayed by an immunofluorescence technique as described by Van dem Borne in 1978. An immunoglobulin M/lambda anti-platelet antibody was found to react in serum as well as in plasma EDTA at room temperature, but not at 37 degrees C. This antibody appeared to be directed against GP78 membrane antigen because this antigen was not detectable by immunofluorescence in platelets collected in EDTA and Na-citrate anticoagulant, whereas a fluorescence signal was revealed in platelets collected in citrate-theophylline-adenosine-dipyridamole. This evidence was confirmed by platelet clumping inhibition tests in which target platelets were pretreated with anti-GP monoclonal antibodies. Clumping in the presence of pseudothrombocytopenia serum was inhibited by anti-GP78kD and anti-GPIIb/IIIa but not by anti-Ib. In this case, GP78 appears to be involved in platelet clumping, together with IIb/IIIa complex. The partial inhibition of the phenomenon observed in citrate-theophylline-adenosine-dipyridamole is probably related to a lower expression of the membrane antigens in platelets collected in this anticoagulant.

Evidence for a relationship between free radicals and insulin action in the elderly.

In forty healthy subjects with normal glucose tolerance divided by age into four groups (group A, subjects with mean age < 25 years [n = 10]; group B, subjects with mean age < 40 years [n = 9]; group C, subjects with mean age < 60 years [n = 11]; group D, subjects with mean age > 75 years [n = 10]) and were matched for body mass index (BMI), lean body mass (LBM), mean arterial blood pressure, and sedentary life style, we determined the plasma O2- production, reduced to oxidized glutathione level ratio (GSH/GSSG), and plasma membrane microviscosity. Euglycemic hyperinsulinemic (1 mU/kg.min-1 for 120 minutes) glucose clamp with simultaneous D-3-H glucose infusion and indirect calorimetry allowed determination of glucose turnover parameters and substrate oxidation. In the oldest group of subjects, a significant increase in plasma O2-production and membrane microviscosity associated with a significative reduction in glucose disappearance rate (Rd), total body glucose disposal (TBGD), and nonoxidative glucose metabolism was found. In group D subjects (n = 10), all of these changes were correlated with one another. In a multiple regression analysis of the pooled data from all study subjects (n = 40), only plasma O2- production levels displayed a statistically significant relation with TBGD and nonoxidative glucose metabolism. In conclusion, in aged patients a significant relationship between free radical production and insulin action seems to exist.

Haemostasis unbalance in Pugh-scored liver cirrhosis: characteristic changes of plasma levels of protein C versus protein S.

Liver cirrhosis leads to a protido-synthetic impairment that alters the levels of blood clotting factors and haemostasis. The aim of this study was to assess the alterations of haemostatic parameters in the evolution of liver cirrhosis scored according to Child's classification, with Pugh's modifications. Thirty-seven patients suffering from alcoholic and non-alcoholic liver cirrhosis, representing stages A5, A6, B7, B8 and C10, were tested for the main blood clotting parameters, i.e. prothrombin time, factor VII, partial activated thromboplastin time, fibrinogen, plasminogen, alpha 2-antiplasmin and physiological inhibitors [antithrombin III (ATIII), protein C (PC), protein S (PS)]. No variations were observed between substages A5 and A6 in any of the parameters, except for coagulation inhibitor levels. Most parameters showed a progressive decrease in stages B and C of the disease. The most significant alterations were found in the physiological coagulation inhibitors, with a sharper decrease in PC and AT III level and a lesser decrease in the level of PS through stages A5 and B8: this evidence could assume an important biological and diagnostic significance.

Plasma GSH/GSSG affects glucose homeostasis in healthy subjects and non-insulin-dependent diabetics.

In healthy subjects (n = 10) and non-insulin-dependent (type II) diabetics (n = 10) matched for age [43.1 +/- 2.2 vs. 41 +/- 4.4 yr, P = not significant (NS)], body mass index (25.1 +/- 1.1 vs. 26 +/- 0.8 kg/m2, P = NS), gender ratio [5 males (M)/5 females (F) vs. 5M/5F], and mean arterial blood pressure (105 +/- 7 vs. 106 +/- 9 mmHg, P = NS), we determined the changes in insulin secretion and action after glutathione infusion (15 mg/min) and the relative increase in the plasma reduced (GSH)/oxidized (GSSG) glutathione ratio. The rise in the plasma GSH/GSSG ratio significantly improved total body glucose disposal in healthy subjects and in diabetic patients. In this latter group, GSH infusion potentiated the beta-cell response to glucose slightly. In controls and diabetics, insulin infusion with a simultaneous increase in the plasma GSH/GSSG ratio significantly enhanced nonoxidative glucose disposal without affecting oxidative glucose metabolism. After glutathione infusion, all metabolic and hormonal changes correlated with a significant decline in plasma membrane microviscosity. In conclusion, the plasma GSH/GSSG ratio seems to play a major role in the modulation of glucose homeostasis mainly in diabetics.

Characterization of hemoglobin lepore variants by advanced mass-spectrometric procedures.

We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.