Insulin and triglyceride levels after different breakfast meal challenges – measurement in dried blood spots (DBS)

I wish to share with my readers an interesting study that the ZRT research team presented recently at the 7th Annual World Congress on Insulin Resistance in San Francisco and won a Bronze Award among appox. two hundred abstracts submitted for competition.

Postprandial insulin and triglyceride (Tg) levels have been suggested as potential clinical tools to help identify individuals at high risk of atherosclerosis and coronary heart disease. Published studies have used a variety of meal types. We investigated the effects of 5 different breakfast meals on postprandial insulin and triglyceride levels, to see if meal type elicited different responses and to help identify a suitable protocol for routine risk assessment.

Blood spots were obtained by finger stick from 19 healthy volunteers after a overnight fast and then 2 hours after eating each of 5 breakfast meals. At least 1 week elapsed between each test meal. After collecting blood spots on a filter paper, they were allowed to dry and stored at room temperature until analysis. 6-mm disks were punched from the dried blood spots into 96-well plates; insulin was assayed using modified serum ELISA assay kits after extraction with extraction buffer, and Tg were assayed using an enzymatic assay following extraction with methanol . The DBS assay correlates highly with simultaneous serum testing, demonstrated in a previously published study. The serum/DBS correlation coefficients for insulin and Tg in that study were r=.93 and r=.91 respectively.

For statistical analysis, insulin and Tg levels were categorized as either normal or abnormal; within the “normal” classification, they were further categorized into “optimal” or “non-optimal”. For insulin, values >15 µIU/mL were “abnormal” and >8 µIU/mL were “non-optimal”, while for Tg, >150 ng/mL was “abnormal” and >100 ng/mL “non-optimal”. Using a Wilcoxon paired sign test, outcomes for each meal were compared to all other meals.

Insulin levels at 2 hours showed a difference in response depending on meal composition. Postprandial insulin appears to correspond to overall carbohydrate content; more abnormal/non-optimal results were seen after meal 4 (highest carbohydrate content) and the fewest abnormal/non-optimal results were seen after meal 2 (lowest carbohydrate content), despite the fact that meal 2 had the highest fat content and the most overall calories. Sustained, higher than optimal insulin levels 2 hours after eating could represent an additional cardiometabolic risk factor, even in individuals whose fasting levels are normal. Dietary choices may therefore increase a person’s chances of postprandial dysmetabolism. In non-diabetics, high postprandial insulin levels are an independent risk factor for coronary artery disease.

Tg levels at 2 hours did not differ significantly between meals, although only about half the values seen were in the normal range. We know from the literature that Tg levels tend to peak around 4 hours postprandially, and in our subjects Tg was still rising after the 2 hour measurement. A 4-hour sample may have elicited a between-meal difference, but compliance issues with collecting a sample 4 hours after eating might preclude postprandial Tg as a routine clinical test.

Convenient, in-home collection of DBS offers a simple tool to research the phenomenon of postprandial dysmetabolism. Assessment of DBS insulin levels 2 hours after a meal may help identify risk in individuals whose fasting levels are normal, and can indicate whether routine dietary choices are exposing them to greater risk than necessary.

Following is the abstract of the study that was presented at the conference.

Insulin and triglyceride levels after different breakfast meal challenges – measurement in dried blood spots (DBS)

Sonia Kapur, Margaret Groves, David Zava, Sanjay Kapur
ZRT Laboratory, Beaverton, Oregon, USA.

The metabolic conditions that predispose individuals to atherosclerosis are thought to be a postprandial phenomenon, termed “postprandial dysmetabolism”. In non-diabetics, high postprandial insulin and triglycerides are independent risk factors for coronary artery disease and cardiovascular events.

Pre- and postprandial serum testing for insulin and triglycerides is inconvenient for patients and practitioners, limiting routine application of these tests and their use in large scale clinical studies.

We developed finger stick DBS tests for insulin and triglycerides that correlate highly with venous serum values (r=.93 and r=.91 respectively), and evaluated these analytes in DBS from 19 healthy volunteers after an overnight fast, before and 2 hours after eating 5 different breakfast meals.

Meals consisted of: 1) glazed donuts, fruit smoothie; 2) boiled eggs, sausages, 2% milk; 3) bagel, cream cheese, boiled egg, 2% milk; 4) pancakes, syrup, tea with cream/sugar; 5) oatmeal, almonds, apple, skim milk.

Postprandial insulin levels >8 µIU/mL were classified as “non-optimal” while >15 µIU/mL were “abnormal”; triglyceride levels >100 ng/mL were “non-optimal” and >150 ng/mL “abnormal”.

For insulin, meal 2 (lowest carbohydrate, highest protein) produced the best (fewer abnormal/non-optimal) postprandial results and meal 4 (highest carbohydrate, lowest protein) the worst (more abnormal/non-optimal) postprandial results. For triglycerides, no meal differed significantly from the others in the number of abnormal or non-optimal classifications.

The convenience of in-home collection and analyte stability offer much wider scale use of the DBS tests for routine clinical assessment and large scale epidemiological studies of postprandial dysmetabolism.

New Definition of Metabolic Syndrome

Did you know the new definition of Metabolic Syndrome? Did you know that waist circumference is now one of five criteria that physicians can use to diagnose metabolic syndrome?

The ATP III guidelines earlier did not consider the waist circumference as an important criteria, to diagnose metabolic syndrome, but a recent statement published online on October 5, 2009 in Circulation actually is a step forward to streamline the use of abdominal obesity in determination of risk of developing metabolic syndrome. The new statement on metabolic syndrome is a combined effort by International Diabetes Federation (IDF), the National Heart, Lung, and Blood Institute (NHLBI), the World Heart Federation, the International Atheroschlerosis Society, and the American Heart Association (AHA).

According to the new definition, people with any three of the following five criteria are considered to have the metabolic syndrome:
1. Increased waist circumference (population and country specific cut points)
2. High triglycerides (≥150 mg/dL)
3. Reduced HDL cholesterol (<40 mg/dL for males and <50 mg/dL for females)
4. Elevated blood pressure (Systolic ≥130 mm Hg and/or diastolic ≥ 85 mm Hg
5. Increased fasting glucose ≥ 100 mg/dL

How is Testosterone related with Cardiometabolic Risks?

Certain risk factors have been shown to cluster with clinical conditions like obesity, type 2 diabetes and cardiovascular problems. Such clustering of risk factors have led investigators to propose a new condition called cardiometabolic risk syndrome. Cardiometabolic risk has gained much attention recently and has been linked directly with visceral adiposity. Few investigations have implicated a direct link between centrally obese men and low levels of testosterone. Association of Testosterone with insulin resistance, obesity, diabetes and atheroschlerosis has been presented earlier but has not received much attention. Higher incidence of metabolic syndrome has also been hypothesized to be associated with decline in testosterone levels. There is evidence that testosterone replacement therapy results in reduced insulin resistance and central obesity in type 2 diabetic men. In this study, we have investigated relationship between testosterone and markers of cardiometabolic risk.

A cohort of male patient samples from ZRT database was selected for the study and categorized in tertiles based on testosterone levels with low (<300),>800). The relationship between testosterone levels and cardiometabolic risk was evaluated using bloodspot testing of insulin, hemoglobin A1c, triglycerides and high sensitivity c-reactive protein. All bloodspot assays were developed in house and showed good correlations with serum/plasma levels of risk markers tested.

A total of 124 male patient samples (41 with testosterone levels <>800 ng/dL) were tested for cardiometabolic risk markers. Mean age (+/- SD) was 50.8 (+/- 12.8). Insulin (4.7 µIU/mL) and high sensitivity c-reactive protein (1.83 mg/L) were found to be significantly lower in the highest tertile with testosterone levels greater than 800 ng/dL when compared with insulin (6.3 µIU/mL) and hs-CRP (3.46 mg/L) concentrations in the lowest tertile with testosterone levels less than 300 ng/dL. There was no significant difference observed in HbA1c and triglyceride levels in different groups.

Our data suggest that a higher testosterone level in men is associated with lower CRP and insulin concentrations. These patients may have a lower risk of developing metabolic syndrome and/or atheroschlerotic cardiovascular events.

Dried Blood Spot Screening for Cardiometabolic Risk Markers shows Benefits of Exercise

The prevalence of diabetes and cardiovascular disease is increasing at an alarming rate. Several clinical and observational studies have demonstrated reduced risk of diabetes when physical activity increases. Simple screening tools are needed to monitor effects of treatment interventions in individuals at high risk.

At ZRT Laboratory in Beaverton, Oregon we assessed the application of dried blood spot technology to measure important cardiometabolic risk markers. Dried blood spot collection has advantages compared to conventional blood draws, such as minimal invasiveness, low sample volume, convenience of repeated measurements and ease of sample storage and transport.

Fifteen participants (28- 63 years of age) enrolled in a fitness study that included 30 minute exercise/ brisk walking five days a week for four months. Levels of insulin, hemoglobin A1c, C-reactive protein and triglycerides were measured in blood spot samples obtained by a simple and easy finger stick before and after the program. Dried blood spot samples were stored at -20C until used for analysis using modified methods developed in house from commercially available assays.

Insulin levels decreased significantly ; triglycerides dropped by 18% and C-reactive protein levels also showed significant improvement. The HbA1c levels remained unchanged during the program.

In conclusion, exercise/ brisk walking for 30 minutes five days a week for four months improved cardiometabolic risk factors independently as confirmed by dried bloodspot testing. This simple screening method has important implications for monitoring overall cardiometabolic health of high risk individuals.