Concepts in bio-molecular spectroscopy: vibrational case studies on metalloenzymes.

Spectroscopic techniques play a major role in the elucidation of structure-function relationships of biological macromolecules. Here we describe an integrated approach for bio-molecular spectroscopy that takes into account the special characteristics of such compounds. The underlying fundamental concepts will be exemplarily illustrated by means of selected case studies on biocatalysts, namely hydrogenase and superoxide reductase. The treatise will be concluded with an overview of challenges and future prospects, laying emphasis on functional dynamics, in vivo studies, and computational spectroscopy.

Reductive activation and structural rearrangement in superoxide reductase: a combined infrared spectroscopic and computational study.

Superoxide reductases (SOR) are a family of non-heme iron enzymes that limit oxidative stress by catalysing the reduction of superoxide to hydrogen peroxide and, thus, represent model systems for the detoxification of reactive oxygen species. In several enzymes of this type, reductive activation of the active site involves the reversible dissociation of a glutamate from the proposed substrate binding site at the iron. In this study we have employed IR spectroscopic and theoretical methods to gain insights into redox-linked structural changes of 1Fe-type superoxide reductases, focusing on the enzyme from the archaeon Ignicoccus hospitalis. Guided by crystal structure data and complemented by spectra calculation for an active site model, the main IR difference signals could be assigned. These signals reflect redox-induced structural changes in the first coordination sphere of the iron centre, adjacent loop and helical regions, and more remote ß-sheets. By comparison with the spectra obtained for the E23A mutant of Ignicoccus hospitalis SOR, it is shown that glutamate E23 dissociates reversibly from the ferrous iron during reductive activation of the wild type enzyme. Moreover, this process is found to trigger a global conformational transition of the protein that is strictly dependent on the presence of E23. Similar concerted structural changes can be inferred from the IR spectra of related SORs such as that from Archaeoglobus fulgidus, indicating a widespread mechanism. A possible functional role of this process in terms of synergistic effects during reductive activation of the homotetrameric enzyme is proposed.

Glycated haemoglobin and the risk of cardiovascular disease, diabetes and all-cause mortality in the Copenhagen City Heart Study.

OBJECTIVE: Individuals with diabetes mellitus (DM) have a considerably elevated risk of developing serious health problems including cardiovascular disease (CVD). Long-term elevated levels of blood glucose in nondiabetic individuals may also be associated with increased risk of CVD. The aim of this study was to investigate the relationships between glycated haemoglobin A(1c) (HbA(1c) ) and CVD, DM and all-cause mortality. SUBJECTS AND DESIGN: The Copenhagen City Heart Study is a prospective study of individuals from the Danish general population. The cohort was followed for 10 years via national registers with respect to incident CVD, DM and all-cause mortality. Follow-up was 100% complete. RESULTS: A total of 5127 subjects were included, of whom 597 had DM. In the nondiabetic population, HbA(1c) was significantly associated with incident CVD events in both univariate [hazard ratio (HR) 1.38, 95% CI 1.11-1.71] and multivariate analyses (HR 1.31, 95% CI 1.05-1.64). In the nondiabetic population, increased levels of HbA(1c) were correlated with developing DM. There was a threefold increase in risk of incident DM per unit increase in HbA(1c) with a univariate HR of 3.83 (95% CI 1.96-7.51). This relationship was essentially unchanged after multivariate adjustments (HR 4.19, 95% CI 2.01-8.71). Furthermore, we found that net reclassification improvement for diagnosed DM and CVD was significantly improved with the addition of HbA(1c) in the analyses. Although not statistically significant, we found a strong trend towards an association between HbA(1c) and all-cause mortality (HR 1.21, 95% CI 0.99-1.47). We did not find the same associations amongst the population with DM. CONCLUSION: In the Danish general population, HbA(1c) was strongly associated with CVD in individuals without DM.

Cardiovascular events in thyroid disease: a population based, prospective study.

No consensus exists whether subclinical thyroid disease should be treated or just observed. Untreated overt thyroid disease is associated with increased risk of cardiovascular disease, and this study was conducted to assess the risk of cardiovascular events in subclinical thyroid disease. The population-based prospective study was conducted in Denmark. A total of 609 subjects from general practice aged 50 years or above with normal left ventricular function were examined. During a median of 5 years of follow-up, major cardiovascular events were documented. In subjects with abnormal TSH at baseline, information about potential thyroid treatment during follow-up was obtained from case reports and mailings. At baseline, 549 (90.7%) were euthyroid (TSH 0.40-4.00 mU/l), 31 (5.1%) were subclinical hypothyroid (TSH>4.00 mU/l), and 25 (4.1%) were subclinical hyperthyroid (TSH<0.40 mU/l). 1 overt hyperthyroid and 3 overt hypothyroid participants were excluded from the analyses. At baseline, the levels of NT-proBNP were inversely associated with the levels of TSH; the lower the levels of TSH, the higher the NT-proBNP concentration. During follow-up, 88 participants died, 81 had a major cardiovascular event, and 28 had a stroke. The incidence of stroke was increased among subjects with subclinical hyperthyroidism, HR 3.39 (95% CI 1.15-10.00, p=0.027) after adjusting for sex, age, and atrial fibrillation. Subclinical hypothyroidism was not related with any of the outcome measurements. Subclinical hyperthyroidism seems to be a risk factor of developing major cardiovascular events, especially stroke in older adults from the general population with normal left ventricular function.

Circulating soluble urokinase plasminogen activator receptor predicts cancer, cardiovascular disease, diabetes and mortality in the general population.

BACKGROUND: Low-grade inflammation is thought to contribute to the development of cardiovascular disease (CVD), type-2 diabetes mellitus (T2D), cancer and mortality. Biomarkers of inflammation may aid in risk prediction and enable early intervention and prevention of disease. OBJECTIVE: The aim of this study was to investigate whether plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) are predictive of disease and mortality in the general population. DESIGN: This was an observational prospective cohort study. Cohort participants were included from June 1993 to December 1994 and followed until the end of 2006. SETTING: General adult Caucasian population. PARTICIPANTS: The MONICA10 study, a population-based cohort recruited from Copenhagen, Denmark, included 2602 individuals aged 41, 51, 61 or 71 years. MEASUREMENTS: Blood samples were analysed for suPAR levels using a commercially available enzyme-linked immunosorbent assay. Risk of cancer (n = 308), CVD (n = 301), T2D (n = 59) and mortality (n = 411) was assessed with a multivariate proportional hazards model using Cox regression. RESULTS: Elevated baseline suPAR level was associated with an increased risk of cancer, CVD, T2D and mortality during follow-up. suPAR was more strongly associated with cancer, CVD and mortality in men than in women, and in younger compared with older individuals. suPAR remained significantly associated with the risk of negative outcome after adjustment for a number of relevant risk factors including C-reactive protein levels. LIMITATION: Further validation in ethnic populations other than Caucasians is needed. CONCLUSION: The stable plasma protein suPAR may be a promising biomarker because of its independent association with incident cancer, CVD, T2D and mortality in the general population.

New risk markers may change the HeartScore risk classification significantly in one-fifth of the population.

The study aim was to determine whether urine albumin/creatinine ratio (UACR), high-sensitivity C-reactive protein (hsCRP) or N-terminal pro-brain natriuretic peptide (Nt-proBNP) added to risk prediction based on HeartScore and history of diabetes or cardiovascular disease. A Danish population sample of 2460 individuals was divided in three groups: 472 subjects receiving cardiovascular medication or having history of diabetes, prior myocardial infarction or stroke, 559 high-risk subjects with a 10-year risk of cardiovascular death above 5% as estimated by HeartScore, and 1429 low-moderate risk subjects with estimated risk below 5%. During the following 9.5 years the composite end point of cardiovascular death, non-fatal myocardial infarction or stroke (CEP) occurred in 204 subjects. CEP was predicted in all three groups by UACR (HRs: 2.1, 2.1 and 2.3 per 10-fold increase, all P<0.001) or by hsCRP (HRs: 1.9, 1.9 and 1.7 per 10-fold increase, all P<0.05), but not by Nt-proBNP (HRs: 1.1, 2.6 and 3.7 per 10-fold increase, last two P<0.001) (P<0.05 for interaction). In the low-moderate risk group, pre-specified gender adjusted (men/women) cutoff values of UACR> or =0.73/1.06 mg mmol(-1) or hsCRP> or =6.0/7.3 mg l(-1) identified a subgroup of 16% who experienced one-third of the CEPs. In the patient group, combined absence of high UACR and high Nt-proBNP> or =110/164 pg ml(-1) (men/women) identified a subgroup of 52% who experienced only 15% of the CEPs. Additional use of UACR and hsCRP in subjects with low-moderate risk and UACR and Nt-proBNP in subjects with known diabetes of cardiovascular disease changed HeartScore risk classification significantly in 19% of the population.

Impact of diabetes on treatment-induced changes in left ventricular structure and function in hypertensive patients with left ventricular hypertrophy. The LIFE study.

BACKGROUND AND AIM: Diabetes is associated with left ventricular hypertrophy (LVH) and impaired systolic function in hypertensive patients, but less is known about its impact on LVH regression and functional improvement during antihypertensive treatment. METHODS AND RESULTS: We performed annual echocardiography in 730 non-diabetic and 93 diabetic patients (aged 55-80 years) with hypertension and electrocardiographic LVH during 4.8-year losartan- or atenolol-based treatment in the Losartan Intervention For Endpoint reduction in hypertension (LIFE) study. Baseline mean blood pressure (BP) and LV mass did not differ between groups. Diabetic patients had higher body mass index and pulse pressure, and lower LV ejection fraction, midwall shortening, stress-corrected midwall shortening, and estimated glomerular filtration rate (all p<0.05), and were more likely to have albuminuria. Despite comparable BP reduction in diabetic and non-diabetic groups during treatment (33/18 vs. 28/16mmHg (ns)), diabetes was associated with higher prevalence of persistent LVH (47 vs. 39%, p<0.05). In multivariate analyses, diabetes independently predicted less LV mass reduction and less improvement in stress-corrected LV midwall shortening (both p<0.01). CONCLUSION: Among hypertensive patients with LVH, diabetes is associated with more residual LVH and less improvement in systolic LV function by echocardiography over 4.8 years of antihypertensive treatment.

Impact of the metabolic syndrome on the predictive values of new risk markers in the general population.

Although the metabolic syndrome (MetS) is positively associated with high-sensitivity C-reactive protein (hsCRP), negatively associated with N-terminal pro-brain natriuretic peptide (Nt-proBNP) and inconsequently related to urine albumin/creatinine ratio (UACR) they are all associated with cardiovascular events. Therefore, we wanted to determine the influence of MetS on the predictive values of UACR, hsCRP and Nt-proBNP. On the basis of the definition of MetS by the International Diabetes Federation, a Danish population sample of 1983 apparently healthy subjects was divided into three groups: 530 subjects without any elements of MetS, 1093 subjects with some elements of MetS and 360 subjects with MetS. During the following 9.5 years the composite end point of cardiovascular death, non-fatal myocardial infarction or stroke (composite cardiovascular end point, CEP) occurred in 204 subjects. In Cox-regression analyses adjusting for age, gender and smoking, all three cardiovascular risk markers predicted CEP independently of MetS. Despite no significant interaction with MetS, high log(hsCRP) was associated with CEP primarily in subjects without any elements of MetS (hazard ratio (HR)=4.5 (1.5-14.0), P<0.01), log(Nt-proBNP) primarily in subjects with some elements of MetS (HR=3.0 (1.6-5.6), P<0.01), and logUACR independently of elements of MetS. Pre-specified gender-adjusted (men/women) cutoff values of hsCRP > or = 6.0/7.3 mg l(-1) predicted CEP in subjects without elements of MetS with positive and predictive values of 11.5 and 98%, respectively. UACR > or = 0.73/1.06 mg mmol(-1) predicted CEP in subjects with MetS with positive and predictive values of 23.5 and 93%, respectively. In apparently healthy subjects, high hsCRP was associated with CEP primarily in subjects without MetS, high Nt-proBNP in subjects with elements of MetS and UACR independently of MetS.

Reversible light-dependent molecular switches on Ag/AgCl nanostructures.

Nanostructured Ag/AgCl substrates were used to generate reversible and highly efficient light-dependent chemical switches based on adsorbed 4,4'-dimercaptoazobenzene (DMAB). DMAB was formed in situ via laser-induced dimerization either from 4-nitrothiophenol (4-NTP) or 4-aminothiophenol (4-ATP). The subsequent reaction pathways of DMAB, however, were quite different as monitored by surface enhanced Raman spectroscopy. In the 4-NTP/DMAB system, AgCl catalyses the reversal of the dimerization. Conversely, irradiation of adsorbed 4-ATP first generated cis-DMAB attached to the surface via two Ag-S bonds, followed by AgCl-catalysed cleavage of one Ag-S bond and cis → trans photoisomerisation of DMAB. In the dark, the trans-isomer thermally reverts to cis-DMAB. The here presented light-dark chemical switches, which work without changing other parameters (e.g., pH, anaerobic vs. aerobic), are based on the (photo)catalytic properties of the Ag/AgCl substrate and do not function on pure metal surfaces.

On the electron transfer mechanism between cytochrome C and metal electrodes. Evidence for dynamic control at short distances.

Cytochrome c was coordinatively bound to self-assembled monolayers of pyridine-terminated alkanethiols on Au and Ag electrodes. The mechanism of heterogeneous electron transfer of the immobilized protein was investigated by cyclic voltammetry and time-resolved surface-enhanced resonance Raman spectroelectrochemistry. The temperature, distance, and overpotential dependencies of the electron transfer rates indicate a change of mechanism from a tunneling controlled reaction at long distances (thicker films) to a solvent/protein friction controlled reaction at smaller distances (thinner films).

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution.

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytic hydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redox transformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric [FeFe] hydrogenase. Through the application of low-temperature resonance Raman spectroscopy, we discovered a novel metastable intermediate that is characterized by an oxidized [FeIFeII] centre and a reduced [4Fe4S]1+ cluster. Based on this unusual configuration, this species is assigned to the first, deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights into the sequence of initial reaction steps, the identification of this species represents a key finding towards the mechanistic understanding of biological hydrogen evolution.

Polyanion binding to cytochrome c probed by resonance Raman spectroscopy.

The interaction of ferricytochrome c with negatively charged heteropolytungstates was studied by resonance Raman spectroscopy. In analogy to previous findings on ferricytochrome c bound to other types of charged interface (Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721, 6722-6728), it was shown that in these complexes the conformational states I and II are stabilized. While in state I, the structure is the same as is in the uncomplexed heme protein, in state II three different coordination configurations coexist, i.e., a six-coordinated low-spin, a five-coordinated high-spin and a six-coordinated high-spin form. These configurations constitute thermal coordination equilibria whose thermodynamic properties were determined. The detailed analysis of the low-frequency resonance Raman spectra reveals that in state II the heme pocket assumes an open structure leading to a significantly higher flexibility of the heme group compared to the native ferricytochrome c. It is concluded that these structural changes are the result of Coulombic attractions between the polyanions and the lysine residues around the exposed heme edge which destabilize the heme crevice. Modifications of these interactions upon variation of the ionic strength, the pH or the type of the polytungstate are sensitively reflected by changes of the coordination equilibria in state II as well as of the conformational equilibrium of state I and state II. The conformational changes in state II significantly differ from those associated with the alkaline transition of ferricytochrome c. However, there are some structural similarities between the acid form of the heme protein stable below pH 2.5 in aqueous solution and the six-coordinated high-spin configuration of the bound ferricytochrome c at neutral pH (state II). This suggests that electrostatic interactions with the heteropolytungstates perturb the ionic equilibria of those amino acid side chains which are involved in the acid-induced transition leading to a significant upshift of the apparent pKa.

Induction of photochemical auto-reduction of cytochrome-c oxidase by an organic peroxide.

Cytochrome-c oxidase aa3 (CcO) from Paracoccus denitrificans interacts with tertiary butyl hydroperoxide (t-Bu-O-O-H, TBHP) by forming an adduct as indicated by an absorption shift at 408/432 nm and the induction of photochemical autoreduction. The adduct was stable at room temperature for several days even under aerobic conditions. Upon irradiation (413 nm) of the adduct, a photoproduct, similar to the oxygenated mixed valence species (607 nm form), was formed, as indicated by the 418/442 and 607 nm signals in the absorption-difference spectrum. It is concluded that the adduct formation changes the photochemical properties of heme a3. A molecular model for the binding mechanism of TBHP to CcO and for the photochemistry of heme a3-TBHP adduct is proposed.

Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study.

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.

Effect of the angiotensin-converting enzyme inhibitor trandolapril on mortality and morbidity in diabetic patients with left ventricular dysfunction after acute myocardial infarction. Trace Study Group.

OBJECTIVES: This study evaluated the efficacy of long-term treatment with the angiotensin-converting enzyme (ACE) inhibitor trandolapril in diabetic patients with left ventricular dysfunction after acute myocardial infarction (AMI). BACKGROUND: Patients with diabetes mellitus have a high mortality following AMI, probably due to a high risk of congestive heart failure and reinfarction. Because ACE inhibition effectively reduces progression of heart failure, it could be particularly beneficial in diabetic patients after AMI. METHODS: The study is a retrospective analysis using data from the Trandolapril Cardiac Evaluation (TRACE) study, which was a randomized, double-blind, placebo-controlled trial of trandolapril in 1,749 patients with AMI and ejection fraction < or =35%. The mean follow-up time was 26 months. RESULTS: A history of diabetes was found in 237 (14%) of the 1,749 patients. Treatment with trandolapril resulted in a relative risk (RR) of death from any cause for the diabetic group of 0.64 (95% confidence interval 0.45 to 0.91) versus 0.82 (0.69 to 0.97) for the nondiabetic group. In the diabetic group, trandolapril reduced the risk of progression to severe heart failure markedly (RR, 0.38 [0.21 to 0.67]), and no significant reduction of this end point was found in the nondiabetic group. CONCLUSIONS: The ACE inhibition after myocardial infarction complicated by left ventricular dysfunction appears to be of considerable importance in patients with diabetes mellitus by saving lives and substantially reducing the risk of progression to severe heart failure.

Antiremodeling effects on the left ventricle during beta-blockade with metoprolol in the treatment of chronic heart failure.

OBJECTIVES: The purpose of the study was to investigate the effects of beta1-blockade on left ventricular (LV) size and function for patients with chronic heart failure. BACKGROUND: Large-scale trials have shown that a marked decrease in mortality can be obtained by treatment of chronic heart failure with beta-adrenergic blocking agents. Possible mechanisms behind this effect remain yet to be fully elucidated, and previous studies have presented insignificant results regarding suspected LV antiremodeling effects. METHODS: In this randomized, placebo-controlled and double-blind substudy to the Metoprolol CR/XL Randomized Intervention Trial in Heart Failure (MERIT-HF), 41 patients were examined with magnetic resonance imaging three times in a six-month period, assessing LV dimensions and function. RESULTS: Decreases in both LV end-diastolic volume index (150 ml/m2 at baseline to 126 ml/m2 after six months, p = 0.007) and LV end-systolic volume index (107 ml/m2 to 81 ml/m2, p = 0.001) were found, whereas LV ejection fraction increased in the metoprolol CR/XL group (29% to 37%, p = 0.005). No significant changes were seen in the placebo group regarding these variables. Left ventricular stroke volume index remained unchanged, whereas LV mass index decreased in both groups (175 g/m2 to 160 g/m2 in the placebo group [p = 0.005] and 179 g/m2 to 164 g/m2 in the metoprolol CR/XL group [p = 0.011). CONCLUSIONS: This study is the first randomized study to demonstrate that the beta1-blocker metoprolol CR/XL has antiremodeling effects on the LV in patients with chronic heart failure and consequently provides an explanation for the highly significant decrease in mortality from worsening heart failure found in the MERIT-HF trial.

Markers of collagen synthesis is related to blood pressure and vascular hypertrophy: a LIFE substudy.

Cardiac fibrosis and high levels of circulating collagen markers has been associated with left ventricular (LV) hypertrophy. However, the relationship to vascular hypertrophy and blood pressure (BP) load is unclear. In 204 patients with essential hypertension and electrocardiographic LV hypertrophy, we measured sitting BP, serum collagen type I carboxy-terminal telopeptide (ICTP) reflecting degradation, procollagen type I carboxy-terminal propeptide (PICP) reflecting synthesis and LV mass by echocardiography after 2 weeks of placebo treatment and after 1 year of antihypertensive treatment with a losartan- or an atenolol-based regimen. Furthermore, we measured intima-media thickness of the common carotid arteries (IMT), minimal forearm vascular resistance (MFVR) by plethysmography and ambulatory 24-h BP in around half of the patients. At baseline, PICP/ICTP was positively related to IMT (r=0.24, P<0.05), MFVR(men) (r=0.35, P<0.01), 24-h systolic BP (r=0.24, P<0.05) and 24-h diastolic BP (r=0.22, P<0.05), but not to LV mass. After 1 year of treatment with reduction in systolic BP (175+/-15 vs 151+/-17 mmHg, P<0.001) and diastolic BP (99+/-8 vs 88+/-9 mmHg, P<0.001), ICTP was unchanged (3.7+/-1.4 vs 3.8+/-1.4 microg/l, NS) while PICP (121+/-39 vs 102+/-29 microg/l, P<0.001) decreased. The reduction in PICP/ICTP was related to the reduction in sitting diastolic BP (r=0.31, P<0.01) and regression of IMT (r=0.37, P<0.05) in patients receiving atenolol and to reduction in heart rate in patients receiving losartan (r=0.30, P<0.01). In conclusion, collagen markers reflecting net synthesis of type I collagen were positively related to vascular hypertrophy and BP load, suggesting that collagen synthesis in the vascular wall is increased in relation to high haemodynamic load in a reversible manner.

Local skin-fold thickness as a clinical predictor of depot size during basal rate infusion of insulin.

OBJECTIVE: To determine the influence of local adiposity on insulin depot size during CSII at basal rate. RESEARCH DESIGN AND METHODS: In 27 diabetic patients, a constant infusion of 125I-labeled Actrapid insulin was given, with U-40 insulin at a rate of 1.12 IU/h in 20 patients, and U-100 at a rate of 1 IU/h in 7 patients. After 16 h of infusion, the steady-state depot size was measured by external counting, and the local skin fold was measured with a Harpenden skin-fold caliper. RESULTS: U-40 insulin infusion resulted in a steady-state depot size of 5.1 U (2.1-10.9 U), and a corresponding skin-fold thickness of 17.8 mm (5-34 mm). A positive correlation was found between depot size and skin-fold thickness. A similar correlation was observed with U-100 insulin. CONCLUSIONS: During basal rate CSII, large variations in local skin-fold thickness create large variations in the steady-state depot size, which is partly predictable just by lifting the skin fold.

The absorption of subcutaneously injected short-acting soluble insulin: influence of injection technique and concentration.

The effect of injection technique on the absorption of subcutaneously injected short-acting insulin [125I-labeled Actrapid (MC), Novo, Copenhagen, Denmark] was investigated in insulin-dependent diabetic patients. In one side of the abdomen insulin was given with a fixed standard technique. In the other side of the abdomen the temperature of the injected insulin, the depth of injection, and the duration of injection were varied. Furthermore, we compared the absorption of U40 and U100 insulin by giving either 8 U of the two insulins or 0.1 ml of both insulins simultaneously to the patients in either side of the abdomen. With regard to the injection technique the only significant finding was a faster absorption rate with deep (12 mm) than with superficial (3 mm) injection. The absorption of U100 insulin was significantly slower than of U40 insulin, when given in the same amount (8 U) as well as in the same volume (0.1 ml).

Absorption of subcutaneously infused insulin: influence of the basal rate pulse interval.

Eight insulin-dependent diabetic patients were given two constant infusions (each 1 IU/h) of 125I-labeled insulin into the abdominal subcutaneous tissue for about 12 h. Insulin was infused in pulses into one side of the abdomen in 6-min intervals (by means of an Auto-Syringe pump) and in the other side of the abdomen, insulin was infused in 1-h intervals (by means of a Medix pump). The size of the subcutaneous depots was continuously measured by counting the radioactivity at the infusion sites. After starting the infusions, the two depots were built up to steady-state levels at the same time and of the same size (approximately 3 IU) and with similar absorption rates. Thus, during basal rate insulin infusion, identical insulin absorption kinetics was achieved, irrespective of a 10-fold difference in the pulse rate.