Comparative Effect of Various Forms of Chromium on Serum Glucose: an Assay for Biologically Active Chromium

This study was undertaken to develop an assay for biologically active chromium suitable for determining the best form of chromium for human supplementation studies. The assay involved monitoring the decrease in fasting serum glucose for 1-3 hours in normal human subjects following ingestion of 100 μg of chromium. The maximum decline occurred 1-2 hours after taking the chromium. The average maximum per cent decrease for 7 subjects was 6.0% for inorganic chromium, 5.7% for conventional brewer’s yeast chromium and 16.8% for a high chromium yeast. A chromium-EDTA complex decreased serum glucose an average of 19.6% in 3 subjects. A placebo yeast and dose-response study demonstrated that the chromium in the high chromium yeast was the factor responsible for the serum glucose lowering effect. The high chromium yeast and CrEDTA had a significantly greater biologically active chromium than inorganic chromium. These findings demonstrate that inorganic chromium is biologically active in man, but chromium in the form of synthetic complexes or high chromium yeast is much more active. Introduction Chromium is recognised as a trace element for both animal and human nutrition. The dietary inorganic chromium must be converted into a biologically active form to function physiologically. This biologically active form was named glucose tolerance factor (GTF) by Mertz [1]. This agent isolated in crude form from brewer’s yeast is able to reverse the impairment of glucose tolerance of chromium-deficient rats overnight. Inorganic chromium is without effect in these rats [2]. Some humans, however, are capable of converting inorganic chromium in vivo into a biologically active form as evidenced by the effectiveness of inorganic chromium in improving the glucose tolerance of three groups: normal infant [3], chromium deficient adults [4] and malnourished infants [5]. The mode of action of biologically active chromium (BAC) is a co-factor of insulin. BAC stimulates the oxidation of glucose in vitro in the presence of insulin, but is ineffective in the absence of insulin [6]. A BAC isolated from brewer’s yeast differs from simple inorganic chromium compounds in rat studies of transport [7]. Thus, it is not surprising that there is no relationship between total chromium content in a diet, food or food extract and biological activity [8,9]. The most widely used assay of BAC is the in vitro insulin-dependent assay of Mertz and Roginski [10] which uses epidymal fat tissue from chromium-deficient rats. This tissue is incubated with different amounts of chromium, insulin and radioactive glucose and the rate of conversion to radioactive carbon dioxide is measured. A potentiation factor is then calculated. Anderson [11] has recently developed a more sensitive assay using adiopocytes from epidermal fat tissue. Mirsky and co-workers [12] have developed a yeast fermentation assay, but it has not been widely used. All these in vitro assays are non-specific as they respond to factors other than chromium. Factors such as pH, osmolarity and compounds such as glucose, glutamate, glutathione and nicotinic acid are known to affect the production of carbon dioxide in the in vitro assay [13-15]. Some foods which contain high concentrations of chromium such as hops [16] actually inhibit insulation potentiation. Also, these in vitro assays are an extract and the activity depends on the pH and solvent used for extraction. The only published in vivo assay involves the injection of chromium samples into mice and monitoring decreases in serum glucose [17]. However, this assay does not respond to inorganic chromium. In addition, the kinetics are widely different for each BAC tested. Nutritional Reports International, 32, (1), 1985. COMPARATIVE EFFECT OF VARIOUS FORMS OF CHROMIUM ON SERUM GLUCOSE: AN ASSAY FOR BIOLOGICALLY ACTIVE CHROMIUM The clinical and public health significance of chromium has been recently reviewed by Mertz [18]. Nutritional risks result from long-term total parenteral alimentation, nutritional formulas and malnourishment. Pregnancy, ageing and diabetes can also increase the risk of chromium deficiency. Chromium deficiency should be considered in all clinical situations where an unexplained insulin resistance develops in patients. Chromium deficiency also influences three recognised risk factors for cardio-vascular disease: impaired glucose tolerance, elevated circulating insulin levels and elevated serum cholesterol. The following research is an effort to develop an in vivo assay for BAC which is applicable for determination of the type of chromium best suited for human supplementation. Materials and Methods Seven normal subjects, 6 males and 1 female, ages 22 to 42 (mean 26 7 years) volunteered to participate in this study with informed consent. Each subject, after an overnight fast of 12 hours, appeared for testing in the morning. A fingerprick sample was taken in the sitting position for the zero hour (baseline) sample. Each subject took one of three forms of chromium. The inorganic form was chromic (III) chloride. The brewer’s yeast form was Formula 350 brewer’s yeast, 1.25 μg chromium/g. This is a brewer’s yeast grown on molasses to which 15% dried yoghurt is added by weight. The high chromium yeast was 2180 μg chromium/g. This yeast was grown in a chromium medium and was hydrolysed during processing. The chromium was taken by all subjects at a dose of 100 μg dissolved in 50 ml of water with the exception of the brewer’s yeast form which required 500 ml of water for a palatable suspension. In addition, a placebo yeast containing < 1 g chromium/g was tested on one subject. A chromium-EDTA complex (CrEDTA) was prepared as described by Gonzalez-Vergara, et. al. [19]. Three subjects ingested 100 g of chromium in this form. A fingerprick sample was taken at 1, 2 and 3 hours post-dose for all forms of chromium. The fingerprick blood samples were taken using an Autclat apparatus, with a Monolet lancet. Blood samples were collected in Microtainer Capillary Blood Serum Separators. The samples were centrifuged at 4°C and the serum kept at 4°C until analysis the same day. Serum glucose was measured by an ultraviolet enzymatic assay kit. Chromium was measured in the commercial products after ashing at 500°C overnight and reconstituted with 0.1 M HNO3 using an atomic spectrophotometer. The actual concentration was very close to the label value. The chromic chloride and CrEDTA were measured in the neutralised solutions and diluted to 100 μg chromium/50 ml water before ingestion. Results The three forms of chromium were consumed by all 7 subjects and the results shown in Table 1. Chromium caused a decrease in serum glucose in all of the 21 experiments. Table: Maximum per cent decrease in Fasting Serum Glucose as a result of ingestion of 100 μg of Chromium. Maximum % Decrease in Serum Glucose Subject Inorganic Brewer’s yeast High chromium yeast 1 3.6 0.6 28.0 2 13.6 10.8 22.7 3 7.4 12.9 17.4 4 4.5 4.8 14.8 5 1.0 1.4 12.5 6 5.3 8.0 9.6 7 6.6 1.2 12.3 Mean S.D. 6.0 ± 3.9% 5.7 ± 5.0% 16.8 ± 6.5% 95% Confidence Interval 2.35 to 9.65% 1.07 to 10.3% 10.7 to 22.8% Nutritional Reports International, 32, (1), 1985. COMPARATIVE EFFECT OF VARIOUS FORMS OF CHROMIUM ON SERUM GLUCOSE: AN ASSAY FOR BIOLOGICALLY ACTIVE CHROMIUM The maximum decrease usually occurred after 1 or 2 hours, but there was variation in the timing from one form of chromium to another and from one subject to another. For all subjects, the decrease in serum glucose was greatest for the high chromium yeast of the three forms of chromium tested. There was no significant difference (p > 0.5) between the serum glucose response for the inorganic chromium and the brewer’s yeast chromium as determined by analysis of variance. The high chromium yeast and inorganic chromium serum glucose response were very highly significantly different (p < 0.005). In addition, the brewer’s yeast and the high chromium yeast were very highly significantly different (p < 0.005). A blank water, placebo yeast and yeast dose-response study were done with one of the subjects and the results are shown in Table 2. Table 2: The maximum per cent Decrease in Serum Glucose as a result of ingestion of Water, Placebo yeast and High Chromium yeast by a single subject. Form Administered Dose of Chromium Maximum % Decrease in Serum Glucose