Plasma Testosterone Level in Male Patients with Metabolic Syndrome

Med. J. Cairo Univ., Vol. 77, No. 1, June: 351-357, 2009 www.medicaljournalofcairouniversity.com Plasma Testosterone Level in Male Patients with Metabolic Syndrome NASHWA EL-SARRAF, M.D. and AMR EL-HADIDY, M.D.* The Departments of Internal Medicine and Critical Care Medicine*, Faculty of Medicine, Cairo University. Abstract Background and Aim: Mild hypogonadism in men have been associated with features of metabolic syndrome. The aim of the present work is to study the role of serum testosterone in middle aged men with metabolic syndrome. Subject and Methods: The study group comprised 50 middle aged men who fulfilled the definition criteria of metabolic syndrome according to WHO definition. They were divided into two groups: Group (1): Included 19 patients with IHD (mean age of 50.6±5.1 years). Group (2): Included 31 patients without IHD (mean age of 50.6±5.1 years). A group of 20 age matched healthy men was used as a control group their age ranged from 41-57 years with a mean of 49.1 ±5.4 years. All patients and controls were subjected to thorough clinical examination including blood pressure, weight, height, BMI and WHR; investigations including ECG, CBC, fasting and postprandial blood sugar, lipid profile and serum uric acid. Total serum testosterone was measured in patients and controls by the use of enzyme chemiluminescent assay on the immulite autoanalyzer. Results: Serum total testosterone level ranged from 0.4-7.8ng/ml in patients group with a mean of 3.8 ± 1.8 while in the control group it ranged from 2.4-9.3ng/ml with a mean of 5.3± 1.9. Comparing serum testosterone level in both groups it was found to be statistically highly significant lower in patients group than in control group p=0.003. A significant negative correlation was found between serum total testosterone level (3.8 ± 1.8ng/ml) and age (50.6±5.1ys) in the the group of patients. r=0.32 p=0.02. A highly significant negative correlation was detected between serum total testosterone level and fasting blood sugar, serum triglycerides, serum total cholesterol, serum uric acid and diastolic blood pressure. ( r=0.728, 0.872, 0.7370, 0.990 and 0.697 respectively). A highly significant positive correlation was detected between serum total testosterone and HDL ( r=0.935) and haemoglobin (r=0.879). Statistically insignificant negative correlation was detected between serum total testosterone level and age, WHR, postprandial blood sugar and LDL. Statistically insignificant positive correlation was detected between serum total testosterone level and BMI. Serum total testosterone level was found to be statistically insignificant lower in group (1) with evidence of IHD (3.45 ± 1.49ng/ml) than in group (2) patients without evidence of IHD (3.97± 1.99ng/ml), p=0.336. Conclusion: We have concluded that middle aged men with metabolic syndrome have lower testosterone level than normal population. There is a possible role of testosterone in the development or progression of metabolic syndrome and/or its components according to WHO definition. Key Words: Testosterone Metabolic syndrome. Introduction MILD hypogonadism in men have been associated with features of the metabolic syndrome, but the association with the metabolic syndrome itself using an accepted definition has been less defined [1]. Metabolic syndrome was initially observed in 1923 by Kyln, who described the clustering of hypertension, hyperglycemia and gout as the syndrome. The WHO definition of metabolic syndrome is: Patients with metabolic syndrome should have one of the following at least: Type 2 diabetes mellitus, impaired glucose tolerance and insulin resistance, plus two of the following at least: Hypertension (blood pressure >140/90mmHg), obesity (body mass index >30Kg/m 2 or waist-hip ratio >0.9 for men and >0.85 for women), hypertriglyceridemia (> 1.7mmol/l) or low serum HDL 351

352 Plasma Testosterone in Metabolic Syndrome level (0.9mmol/l for men and <1mmol/l for women) and microalbuminuria (albumin-creatinine ratio >2.5mg/mmol for men and >3.5mg/mmol for women) [2]. The third Report of the National Cholesterol Education Program (NCEP), Expert Panel on Detection, Evaluation and Treatment of high blood cholesterol in adults (Adult Treatment Panel III) (ATP III) proposed the most widely used criteria for diagnosis of metabolic syndrome. According to the ATP III criteria, the metabolic syndrome is identified by the presence of three or more of the following: 1- Central obesity as measured by waist circumference: Men >102cm, women >88cm. 2-Fasting HDL cholesterol: Men <40mg/dl, women <50mg/dl. 3- Fasting blood triglycerides >150mg/dl. 4- Blood pressure >130/85mmHg. 5- Fasting blood glucose >1 10mg/dl [3]. The international Diabetes Federation (IDF) in 2005 proposed this definition for metabolic syndrome; central obesity (waist circumference >94 cm for European men and >80cm for European women) with ethnicity specific values for other groups plus two of the following: 1- Blood triglycerides level >150mg/dl or specific treatment for this lipid abnormality. 2- HDL cholesterol <40mg/dl in males and <50mg/dl in females or specific treatment for this lipid abnormality. 3- Raised blood pressure: Systolic blood pressure >130 or diastolic blood pressure >85mmHg or treatment of previously diagnosed hypertension. 4- Fasting plasma glucose >1 00mg/dl or previously diagnosed type 2 diabetes mellitus. Cook and colleagues in 2003 showed that the prevalence of the metabolic syndrome in children was approximately 6 to 7%. Prevalence of the metabolic syndrome in older individuals was 28.1% by ATPIII criteria and 21% by WHO criteria [4]. Endogenous hyperinsulinemia(ehi) is associated with metabolic syndrome, insulin resistance and early type 2 diabetes. Additionally, ehi is associated with hypertension and atheroscleropathy (coronary artery disease). Endogenous hyperinsulinemia is also associated with elevated FFA, PAI-1, elevated sympathetic tone and activity and increased sodium and water reabsorption leading to volume expansion which leads to and supports hypertension. Insulin, proinsulin and amylin have been noted to contribute to elevation of angiotensin II with increases in renin and aldosterone [5]. Obesity is associated with profound alterations in androgen secretion, transport, metabolism and action, according to a dichotomous behavior depending on sex. Obese men are characterized by a progressive decrease of testosterone levels with increasing body weight. Moreover there are theoretical possibilities that low testosterone in men and high free testosterone fraction in women play a role in the development of the metabolic syndrome [6]. The aim of the present work is to study the role of serum testosterone in middle aged men with metabolic syndrome. Subjects and Methods Subjects: This study comprised 50 middle-aged men, their ages ranged from 41-59 years with a mean of 50.6±5.1, who fulfilled the definition criteria of metabolic syndrome according to WHO definition. A group of 20 age-matched healthy men was used as a control group, their ages ranged from 41-57 years with a mean of 49.1 ±5.4. An informed consent was taken from patients and controls. Methods: All patients and controls were subjected to the following: A- Complete medical history taking with special emphasis on: Age. Hypertension. Diabetes mellitus. Dyslipidemia. Ischemic heart disease. B- Thorough clinical examination with special emphasis on: Blood pressure Height, weight, Body Mass Index (BMI). Waist circumference, Hip circumference, Waist/Hip ratio (WHR). Cardiac examination.

Nashwa El-Sarraf & Amr El-Hadidy 353 C- Investigations: 1- Electrocardiogram: Standard 12 lead ECG was done and analyzed for evidence of ischemic heart disease. 2- Laboratory tests: Complete blood picture. Fasting and post prandial blood sugar. Lipid profile: a- Serum total cholesterol. b- Serum triglyceride. c- HDL-cholesterol. d- LDL-cholesterol. Serum uric acid. Serum total testosterone: Was measured by the use of enzyme chemiluminescent assay on the immulite autoanalyzer. D- Statistical methodology: Analysis of data was performed using SPSS 10 windows package as follows: Description of quantitative variables in the form of range, mean and standard deviation. Mean = sumx/n SD = Sumx2-sumx2/m n-1 Description of quantitative variables in the form of frequency and percentages. Chi-square test was used to compare qualitative groups. Chi-square = (observed-expected)2 expected t-student test of two independent samples was used to compare two groups as regard quantitative variable. One way ANOVA test was used to compare more than two groups as regard quantitative variable. r-test (correlation coefficient) to rank different parameters again each other either directly or indirectly. p-value. p>0.05 insignificant. p<0.05 significant. p< highly significant. Results Clinical and laboratory data of cases and control groups (Table 1). Table (1): t Test showing clinical and laboratory data of cases and control groups. Parameter Cases Control Range Mean ± SD Range Mean ± SD p.value Age (ys) 41-59 50.6±5.1 41-57 49.1 ±5.4 0.268 BMI 30-33.5 30.5±0.7 23.1-27.5 25.1 ± 1.1 WHR (kg/m 2 ) 0.91-0.97 0.93±0.02 0.83-90 0.88±0.02 0.000 HB (gm/dl) 11-14 12.1 ±0.8 13-14 13.1 ±0.4 0.002 FBS 110-130 118.4±3.9 81-99 90.4±5.1 0.000 PPBS 140-200 159.9± 10.0 105-118 112.5±3.9 TG 153-190 165±8.1 130-147 140.7±4.5 HDL 30-39 34.4±2.6 41-59 49.9±4.9 0.002 Total cholesterol 205-248 224.6± 10.02 174-198 185.9±5.9 0.000 Uric acid 4-8.2 6.4± 1.2 3.1-5.1 3.9±0.6 Total Testosterone (ng/ml) 0.4-7.8 3.8± 1.8 2.4-9.3 5.3± 1.9 0.003 Comparing body mass index (BMI) in both groups, it ranged from 30-33.5Kg/m 2 in the group of 50 patients with a mean of 30.5 ±0.7, while in the control group it ranged from 23.1-27.5Kg/m 2 with a mean of 25.1 ± 1.1. BMI was found to be statistically highly significant higher in patients as compared to control group. p= (Table 1). As regarding, waist hip ratio (WHR), it ranged from 0.91-0.97 in the group of patients with a mean of 0.93±0.02, while in the control group it ranged from 0.83-0.90 with a mean of 0.88±0.02. So WHR was found to be statistically highly significant, higher in patients group than in control group. p=0.000 (Table 1).

354 Plasma Testosterone in Metabolic Syndrome Hypertension (HTN) was detected in patients, the diastolic blood pressure ranged from 95-110mmHg with a mean of 99.8 ±4.03. Ischemic heart disease (IHD) was detected in 19 patients out of 50 patients (38%) (group 1). Comparing haemoglobin level (HB) in both groups, it ranged from 11-14gm/dl in the group of patients with a mean of 12.1 ±0.8, while in the control group, it ranged from 13-14gm/dl with a mean of 13.1 ±0.4. Thus HB was found to be statistically highly significant lower in patients than in control group p=0.002 (Table 1). Fasting blood sugar (FBS) ranged from 110-130mg/dl in patients group with a mean of 118.4±3.9 while in control group, it ranged from 81-99mg/dl; with a mean of 90.4 ±5.1. Comparing FBS in both groups, it was found to be statistically highly significant higher in patients group than in control group p=0.000 (Table 1). Regarding post prandial blood sugar (PPBS) it ranged from 40-200mg/dl in patients group with a mean or 159.9 ± 10.0 while in control group, it ranged from 105-118mg/dl with a mean of 12.5 ± 3.9. So PPBS was found to be statistically highly significant more in patients group than in control group p= (Table 1). Comparing serum triglycerides (TG) in both groups, it ranged from 153-190mg/dl in patients group with a mean of 165 ±8.1 while in the control group, it ranged from 130-147mg/dl with a mean of 140.7±4.5. Serum triglycerides level was found to be statistically highly significant greater in patients group than in control group p= (Table 1). As regard, high density lipoprotein (HDL), it ranged from 30-39mg/dl in patients group with a mean of 34.4±2.6 while in control group it ranged from 41-59mg/dl with a mean of 49.9 ±4.9. So HDL was found to be statistically highly sigl 1 ificant lower in patients group than in control group p=0.002 (Table 1). Serum total cholesterol ranged from 205-248mg /dl in patients group with a mean of 224.6 ± 10.02, while in control group it ranged from 174-198mg/dl with a mean of 185.9±5.9. Total cholesterol was found to be statistically highly significant more in patients group than in control group p=0.000 (Table 1). Comparing serum uric acid level in both groups, it ranged from 4-8.2mg/dl in patients group with a mean of 6.4± 1.2, while in control group it ranged from 3.1-5.1mg/dl with a mean of 3.9 ±0.6. Thus it was found to be statistically highly significant more in patients group than in control group p= (Table 1). Serum total testosterone level ranged from 0.4-7.8ng/ml in patients group with a mean of 3.8 ± 1.8, while in control group it ranged from 2.4-9.3ng/ml with a mean of 5.3 ± 1.9. Comparing serum total testosterone level in both groups it was found to be statistically highly significant lower in patients group than in control group p=0.003 (Table 1). Serum total testosterone level was 28% lower in the 50 patients with metabolic syndrome as defined by WHO than control group without metabolic syndrome. 6 5 4 3 2 1 0 3.8 Cases 5.8 Controls Fig. (1): Serum total testosterone level among both studied groups. Table (2): Correlations between total testosterone and all parameters in 50 patients group. Age (ys) WHR BMI (kg/m 2 ) HB% (gm/dl) FBS PPBS TG HDL CHOL Uric Acid Diast BP (mmhg) Mean ± SD 50.6± 0.93± 30.5± 12.1± 118.4± 159.9± 165± 34.4± 224.6± 6.4± 99.8± 5.1 0.02 0.8 0.8 3.9 10 8.1 2.6 10.02 1.2 4.04 Pearson correlation p-value 0.32 0.02 0.05 0.717 0.992 0.879 0.728 0.000 0.249 0.081 0.872 0.002 0.935 0.737 0.003 0.990 0.697

Nashwa El-Sarraf & Amr El-Hadidy 355 Significant negative correlation was found between serum total testosterone level (3.8 ± 1.8ng/ml) and age (50.6 ±5.1 ys) in the group of patients r=0.32, p=0.02. Statistically insignificant negative correlation was detected between serum total testosterone level (3.8± 1.8ng/ml) and WHR (0.93 ±0.02) r= 0.05, p=0.717. Comparing serum total testosterone level (3.8 ± 1.8ng/ml) and BM I (30.5 ±0.7Kg/m 2 ) statistically insignificant positive correlation was detected r=, p=0.992. Serum total testosterone level (3.8 ±.8ng/ml) was negatively correlated with diastolic blood pressure (99.8 ±4.04mm Hg) and this negative correlation was statistically highly significant r= 0.697, p=. level (3.8± 1.8ng/ml) and serum total cholesterol level (224.6± 10.02mg/dl) r=0.737, p=0.003. As regard HDL, there was statistically highly significant positive correlation between HDL (34.4 ±2.6mg/dl) and serum total testosterone level (3.8± 1.8ng/ml). r=0.935, p=. On the other hand, comparing serum total testosterone level (3.8 ± 1.8ng/ml) and LDL (158.14 ± 11.45mg/dl), statistically insignificant negative correlation was detected r= 0.16, p=0.14. Comparing serum total testosterone level (3.8 ± 1.8ng/ml) and serum uric acid level (6.4± 1.2mg/dl), a statistically highly significant negative correlation was detected r= 0.990, p=. 7 6 6.8 120 100 80 99.8 5 4 3 2 3.8 60 1 40 20 0 3.8 Testosterone Diastolic Fig. (2): Mean serum total testosterone level and diastolic blood pressure. Statistically highly significant positive correlation was detected between serum total testosterone level (3.8 ± 1.8ng/ml) and haemoglobin (12.1 ± 0.8gm/dl) r=0.879, p=. There was stastically highly significant negative correlation between serum total testosterone (3.8± 1.8ng/ml) and FBS level (118.4 ±3.9mg/dl) r= 0.728, p=0.000. On the other hand, comparing serum total testosterone level (3.8 ± 1.8 ng/ml) and PPBS (159.9 ± 10mg/dl) a statisticaily insignificant. Negative correlation was detected r= 0.249, p=0.081. Serum total testosterone level (3.8 ± 1.8ng/ml) was found to be negatively correlated with triglycerides level with a high statistical significance (165±8.1mg/dl) r= 0.872, p=0.002). Statistically highly significant negative correlation was detected between serum total testosterone 0 Testosterone Uric acid Fig. (3): Mean serum total testosterone level and serum uric acid level. IHD was detected in 19 out of 50 patients (Table 3). The patients could be classified into a group of 19 patients with evidence of IHD and a group of 31 patients without evidence of IHD (Table 3). Serum total testosterone level was found to be statistically insignificant. Lower in the group or 19 patients with evidence of IHD (3.45 ± 1.49ng/ml) than in the group of 31 patients without evidence of IHD (3.97±l.99ng/ml), p=0.336 (Table 3). Table (3): Total testosterone level in IHD and non IHD patients. IHD N Range Mean ± p SD value Total Testosterone Absent 31 0.4-7.8 3.97± 1.99 0.336 Total Testosterone Present 19 1.1-5.8 3.45± 1.49 0.336 Discussion Mild hypogonadism in men have been associated with features of the metabolic syndrome, but the association with the metabolic syndrome itself using an accepted definition has been less defined [1].

356 Plasma Testosterone in Metabolic Syndrome Low levels of male sex hormones in men [1], while high free testosterone in women [6], have been associated with components of the metabolic syndrome. In this study, the testosterone level which was measured in 50 middle-aged men who fulfilled the definition criteria of metabolic syndrome as defined by WHO was significantly lower in patients Vs controls p=0.003. Serum total testosterone was 28% lower in patients Vs control group in comparison to another study which revealed serum total testosterone 19% lower in 345 patients with metabolic syndrome vs controls p value < [9]. Testosterone itself may have a causal role in the pathogenesis of the metabolic syndrome or its components by increasing muscle mass, decreasing abdominal obesity and improving insulin sensitivity [7]. Higher BMI was detected in patients with metabolic syndrome in our study in comparison to controls (p=) as well as higher WHR (p 0.000). These results are supported by previous studies, one of these studies is a population-based study by Laaksonen and his colleagues in 2003 [9]. Overall abdominal obesity increases glucocorticoids turnover and production, resulting in abnormal regulation of the hypothalamic-pituitary-adrenal axis and possible mild hypogonadism in men [8]. Serum total testosterone level is insignificant negatively correlated with WHR matching with results given by [1]. In this work, patients with metabolic syndrome had hypertension with diastolic blood pressure (99.8±4.04mmHg) matching with results of previous study [9] and a statistically highly significant negative correlation was detected between serum total testosterone level and diastolic blood pressure, r=0.697, p= as had been reported by [10]. Hypertension is one of most important components of metabolic syndrome, the main etiology of metabolic syndrome is supposed to be due to insulin resistance. Insulin resistance/hyperinsulinemia induces blood pressure elevation by sodium retention, activation of sympathetic nervous system and renin-angiotensin system (RAS) and promotion of vascular cell growth. Moreover, activated RAS actually angiotensin II and salt intake lead to insulin resistance by inhibiting insulin signaling [11]. The 50 men with metabolic syndrome in this work had lower hemoglobin level than controls p=0.002 in agreement with [12]. Serum total testosterone level in patients group showed statistically highly significant positive correlation with hemoglobin level r=0.879. The same results were given by Bhasin and his colleagues in 2001 [12]. Testosterone regulates erythropioesis through its effects on erythropoietin and stem cell proliferation. In this study and in agreement with [9], patients with metabolic syndrome had higher FBS and PPBS when compared to controls. There was statistically highly significant negative correlation between serum total testosterone and FBS, r=0.728, this was consistent with results given by [9]. On the other hand, insignificant negative correlation was detected between serum total testosterone and PPBS, in contrast to results of [9] which revealed statistically significant negative correlation between serum total testosterone level and PPBS, p<. This may be attributed to larger number of patients included in their study. In previous cross-sectional studies, low concentrations of testosterone and sex hormone-binding globulin (SHBG) have been associated with visceral obesity, insulin resistance or hyperinsulinemia and dyslipidemia [13]. Serum triglycerides and total cholesterol were detected to be significantly higher in our 50 men with metabolic syndrome while LDL were insignificantly higher in our 50 patients. Serum total testosterone was found to be negatively correlated with triglycerides, total cholesterol and LDL, r=0.872, 0.737 and 0.16 respectively. On the other hand, significant positive correlation was detected between serum total testosterone and HDL. r=0.935. This is consistent with results given by [9]. The association of testosterone and SHBG with an altered lipid profile is partly secondary to abdominal fat accumulation but there also appears to be an independent relationship between low level of testosterone and hyperinsulinemia [1] and dyslipidemia [14]. Serum uric acid level was higher in patients with metabolic syndrome and statistically highly significant negative correlation was detected with serum total testosterone level r=0.99. This is consistent with results reported by [15]. Hyperuricemia induced by hypouricosuria often accompanies the metabolic syndrome and insulin resistance has been hypothesized as the common underlying defect [15]. There is statistically significant negative correlation between serum total testosterone level (3.8± 1.8ng/ml) and age of 50 patients with metabolic syndrome (50.58 ±5.1 years) r=0.032. Aging is accompanied by insulin resistance and a decline in testosterone secretion [16].

Nashwa El-Sarraf & Amr El-Hadidy 357 IHD was detected in 19 out of 50 patients (38%) which is consistent with results reported by Haffner and his colleagues [14]. Serum total testosterone level was found to be statistically insignificant lower in the group of 19 patients with evidence of IHD than in the group of 31 patients without evidence of IHD. Low testosterone levels may predispose to visceral obesity, leading to dysregulation of fatty acid metabolism, which in turn promotes insulin resistance [17]. Insulin resistance is frequently observed in obese subjects has been established as an independent risk factor for development of both type 2 diabetes and coronary artery disease [18]. In conclusion: Middle-aged men with evidence of metabolic syndrome have lower total testosterone level than normal population. There is a possible role of testosterone in the development and/or progression of the metabolic syndrome and/or its components according to WHO definition. Large scale studies are recommended in different age groups. References 1- PHILLIPS G.B., JING T. and HEYMSFIELD S.B.: Relationships in men of sex hormones, insulin, adiposity and risk factors for myocardial infarction. Metabolism, 52: 784-790, 2003. 2- SHAW J.E. and CHISHOLM D.J.: Epidemiology and prevention of type 2 diabetes and the metabolic syndrome. emja, 179 (7): 379-383, 2003. 3- American Heart Association, 2004. 4- SCUTERI A., NAJJAR S.S., MORRELL C.H., et al.: The metabolic syndrome in older individuals: Prevalence and prediction of cardiovascular event the Cardiovascular Health Study. Diabetes Care, 28 (4): 882-887, 2005. 5- YUDKIN J.S., MAY M., ELWOOD P., et al.: Concentrations of proinsulin like molecules predict coronary heart disease risk independently of insulin: Prospective data from the Caerphilly Study. Diabetology, 45: 327-336, 2002. 6- PASQUALI R: Obesity and androgen: Facts and perspectives. Fertil Steril, 85 (5): 1319-1340, 2006. 7- MARIN P., HOLMANG S., JONSSON L., et al.: The effect of testosterone treatment on body composition and metabolism in middle-aged obese men. International Journal of obesity and Related Metabolic Disorders, 16: 991-997, 1992. 8- WALKER B.R.: Steroid metabolism in metabolic syndrome X. Best Practice and Research in Clinical Endocrinology and Metabolism, 15: 111-122, 2001. 9- LAAKSONEN D.E., LEO NISKANEN, KARI PUNNON- EN, et al.: Sex hormones, inflammation and the metabolic syndrome: A population based study. European Journal of Endocrinology, 149: 601-608, 2003. 10- KHAW K.T. and BARRETT-CONNOR E.: Blood Pressure and endogenous testosterone in men: An inverse relationship. Journal of Hypertension, 6: 329-332,1988. 11- KAMIDE K. and RAKUGI H.: Hypertension. Nippon Rinsho., 62 (6): 1104-1107, 2004. 12- BHASINS, WOODHOTASC I., CASABURI R., et al.: Testosterone dose response relationships in healthy young men. American Journal of physiology, Endocrinology and Metabolism, 281: E1172-E1181, 2001. 13- SIMON D., PREZIOSI P., BARRETT-CONNOR E., et al.: Interrelation between plasma testosterone and plasma insulin in healthy adult men: The Telecom. Study. Diabetologia, 35: 173-177, 1992. 14- HAFFNER S.M., MYKKANEN L., VALDEZ R.A., et al.: Relationship of sex hormones to lipids and lipoproteins in nondiabetic men. Journal of Clinical Endocrinology and Metabolism, 77: 1610-1615, 1993. 15- MARANGELLA M.: Uric acid elimination in the urine. Pathophysiological implications. Contrib. Nephrol., 147: 132-148, 2005. 16- FELDMAN H.A., LONGCOPE C., DERBY C.A., et al.: Age trends in the level of serum testosterone and other hormones in middle-aged men: Longitudinal results from the Massachustts male aging. J. Clin. Endocrinol. Metab., 87: 589-598, 2002. 17- BODEN G., JADALI F., WHITE J., et al.: Effects of fat on insulin-stimulated carbohydrate metabolism in normal men J. Clin. Invest, 88: 960-966, 1991. 18- REAVEN G.M.: Multiple CHD risk factors in type 2 diabetes: beyond hyperglycaemia. Diabetes Obese. Metab., 4 (Suppl. 1): S13-8, 2002.