Can anthropometric measurements and diet analysis serve as useful tools to determine risk factors for insulin-resistant diabetes type 2 among white and black Americans?

OBJECTIVE: Central obesity is implicated in the development of insulin resistance by increasing insulin demand and eventually leading to hyperinsulinemia. Anthropometric measurements have been helpful in determining the risk factors in developing diabetes mellitus type 2. In this study we investigated whether anthropometric measurements differ among diabetics of different races. We also evaluated whether nutrient intake of these individuals was related to anthropometric measurement changes. METHODS: Subjects were recruited from four groups: white control (n = 10), black control (n = 10), white diabetic (n = 5), and black diabetic (n = 10). The diabetic subjects had type 2 diabetes with insulin resistance on insulin monotherapy (age and sex matched). The following determinations were made: diet analysis, body mass index (kg/m(2)), the ratio of waist (umbilical level) to hip (maximum at buttocks) circumference, the ratio of waist to thigh (mid-thigh), and body fat percentage. RESULTS: The micronutrient consumption was fairly similar in all groups with the exception of vitamin A (greatest consumption in the white control group, P < 0.05; and the lowest consumption in the black control group, P < 0.05). The data also suggested that central obesity (greatest waist-to-hip ratio) was present in the individuals with type 2 diabetes. The higher total fat, including saturated, monounsaturated, polyunsaturated, and cholesterol, intake in the diabetic groups were observed. CONCLUSION: The type of fat consumed may be as important as the total fat consumption in the development of insulin resistance. The diet analysis can provide valuable information about the dietary habits of an individual and the possible causes of metabolic problems leading to a disease state. However, genetic factors must be considered when looking at diabetes incidence in different ethnic groups. For example, even though the black diabetic group consumed less fat in comparison with the other groups, their body fat percentages were higher. Therefore, we cannot conclude that high fat intake is primarily responsible for increased body fat percentage. Although anthropometric measurements are a useful tool in risk assessment, researchers should consider anatomic differences among different racial groups as covariables. Diet analysis when used in conjunction with anthropometric measurements can serve as a useful tool to detect whether metabolic alterations are related to dietary habits.

Factors influencing maternal behavior in the hubb/hubb mutant mouse.

We examined the maternal behavior of hubb/hubb mutant mice and normal control (+/hubb) siblings. From previous observations we noted that mutants groom their pups less, suckle less than normal, and often cannibalize the young. To date, these observations had not been quantified. Although prolactin (PRL) is linked to maternal behavior, it was difficult to measure because of the hyperirratibility of the mutant mice. Consequently, dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), were measured in the median eminence in brains of both normal and mutant mice. Tyrosine hydroxylase, the rate-determining step in dopamine synthesis, was localized in the brain by immunohistochemistry. Five mutant and nine normal dams were observed for pup retrieval and crouching. Mean time for pup retrieval was slower (p < 0.06) for mutants (28.09 s) than for normal dams (18.49 s). Crouching was the same for both strains. Mutant pups were cold to the touch, and not well groomed. Brains from both strains were examined at Day 11 and Day 18 of gestation and Day 2 and Day 11 of lactation. Qualitatively, tyrosine hydroxylase localization in the arcuate nucleus and median eminence was the same in both strains for the gestation samples. The decrease in staining observed from gestation to lactation in the normal mice was increased in the mutants. Dopamine was similar in both strains at all stages, but DOPAC was significantly higher at early lactation in the mutants. We do not assume an absolute inverse relationship between dopaminergic activities and prolactin, but it is likely that the increase in DOPAC in the mutant reflects a decrease in prolactin, which could contribute to the diminished maternal care in the mutants.

Isolation and partial polypeptide characterization of bovine neutrophil plasma membranes.

The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3 alpha-6 alpha-diphenylglycouril, and proteins were separated by SDS-PAGE and autoradiographed. Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5'-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for beta-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 micrograms/10(9) bovine blood neutrophils. The SDS-PAGE of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.

Effect of dietary calcium concentrations during gestation and lactation in the sow on milk composition and litter growth.

This study evaluated the effects of diets containing varying amounts of Ca on milk composition and litter characteristics of sows. Yorkshire sows were fed one of the following diets: low Ca (.38%; n = 5), standard Ca (.75%; n = 5), or high Ca (1.12%; n = 4). Diets were fed from d 42 of gestation to d 42 of lactation. Milk was collected on d 7 +/- 1, 21 +/- 1, and 42 +/- 1 of lactation. Lactose was greater (6.4%, P < .05) in milk from sows fed low Ca than in those fed standard (5.5%) or high (5.8%) Ca, but only on d 7 of lactation. No treatment effect or time x treatment interaction was detected for total protein or casein concentrations. Calcium increased (P < .05) over time irrespective of treatment. There was an increase (P < .05) in ADG on d 7 in pigs from high-fed (.28 kg) and low-fed sows (.20 kg) compared with those from standard-fed sows (.13 kg). In conclusion, body Ca is so physiologically regulated that minor dietary alterations have little overall effect on milk composition. Alterations in ADG for pigs, especially from sows fed high Ca, warrant further investigation.

Modulation of bovine neutrophil functions by monoclonal antibodies.

The main objective of the study reported here was to generate a panel of monoclonal antibodies (MAB) to bovine neutrophil surface antigens, and to identify MAB that modulate neutrophil chemotaxis, respiratory burst, and phagocytosis. A further objective was to study MAB reactivity with resting and activated neutrophils, to identify activation antigens and adhesion molecules. A panel of 14 MAB was generated by producing murine hybridomas. Neutrophils incubated with MAB at 4 C for 2 hours were used in chemotaxis, respiratory burst, and phagocytosis assays. Chemotaxis was evaluated in Boyden chambers, using Escherichia coli endotoxin-activated fetal bovine serum as the chemoattractant. Respiratory burst was determined by measuring chemoluminescence of neutrophils incubated with 5-amino-2,3-dihydro-1,4-phthalazinedione, and serum opsonized zymosan. Phagocytosis was determined by flow cytometry, using fluorescein-labeled Staphylococcus aureus. The MAB S7G8, S5F8G10, S7E10, and S5F8B8 enhanced chemotaxis (to > 125% of control). The MAB S7E10 and S8D9 enhanced respiratory burst activity (to > 125% of control), whereas MAB S2G8, S4G10, S8G10, and S5F8B8 caused inhibition (to < 75% of control). The MAB S2G8, S4G10, S8G10, and S5F8G10 enhanced phagocytosis (to > 125% of control). Chemotaxis, respiratory burst, and phagocytosis values of neutrophils not bound with MAB served as controls for comparison. The MAB binding for nonactivated neutrophils (at 4 C) ranged from 9 to 100%, and for activated neutrophils (at 37 C; at 37 C with phorbol myristate acetate) from 90 to 100%. Binding of MAB S4F5, S5F8B8, S6C6, S7E10, S8D9, and S5F8G10 increased when neutrophils had been incubated at 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophil surface antigens, and monoclonal antibody production and characterization.

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surface antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (MAB) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by whole-cell ELISA. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by MAB was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I. The flow cytometric method was proven to be more sensitive and rapid than ELISA to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 x 10(5) neutrophils and 1 micrograms of fluorescein-labeled F(ab')2/assay as the second antibody. The optimal conditions for hybridoma screening by ELISA were neutrophil concentration of 2.5 x 10(5)/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromatogenic substrate. Tissue culture plates as solid support and 3,3', 5,5'-tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive. Panel MAB reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter MAB reactivity. Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with 125I. Monoclonal antibody S7G8 identified a 65-kd protein and MAB S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, MAB S7G8 and S8G10 are comparable to human CD15, CD16, and CD64 molecules. The MAB generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.

Flow cytometric study of oxidative burst activity in bovine neutrophils.

A flow cytometric procedure was evaluated to measure the oxidative burst activity (hydrogen peroxide formation) of bovine neutrophils. The method involves measuring the oxidation of intracellular dichlorofluorescein to fluorescent dichlorofluorescein (DCF). Phorbol myristate acetate (PMA) was used to perturb the neutrophil plasma membrane. The sources of variation introduced into the DCF assay were also examined. The sources of variation were attributable to the isolation of neutrophils from blood, variation between duplicate assays and duplicate flow cytometric determinations of oxidative product formation, variation in neutrophil oxidative product formation among cows, and the variation (over repeated daily and weekly neutrophil isolations) in neutrophil oxidative product formation. A final objective was to determine effects of dexamethasone on oxidative product formation, and whether differences existed between blood and mammary neutrophils in oxidative product formation. There was an increasing trend in the formation of DCF with increasing time of incubation and with increasing PMA concentration. Increasing the concentration of PMA decreased lag time and increased the rate of oxidative product formation. The increase in DCF formation was statistically significant up to a PMA concentration of 10 ng/ml. This concentration was considered optimal for bovine neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian LHRH during embryonic development: measurement by competitive ELISA with a monoclonal antibody.

A mouse monoclonal antibody directed against chicken Gln8-luteinizing hormone-releasing hormone (cLHRH-I) was developed and characterized. This antibody was used for the development of a competitive microtiter plate enzyme-linked immunosorbent assay for avian LHRH. The assay was validated for use with tissue and was used at a working range between 5 pg and 10 ng per sample. Using this procedure, cLHRH-I and II were assayed in whole brain extracts of Japanese Quail embryos. Samples were taken at regular intervals between Day 6 of incubation through Day 1 posthatch. There were 10 samples taken at each age with 2 replicates of the entire sampling regime. Data from males and females were pooled. LHRH concentrations were low, then rose to higher levels (15 pg/mg tissue) between Days 10 through 13 and decreased thereafter. These changes are likely to be correlated with the activation of the hypothalamic-pituitary-gonadal axis. This is particularly apparent in later embryonic development.

Calcium and calmodulin levels in late embryonic and early hatched Japanese quail brain.

Characterizing the last phases of embryonic avian brain development are increased brain activity and increased absorption of shell calcium. The calcium-binding protein, calmodulin, regulates many activities of calcium. In neural tissue, calmodulin modulates neural transmission, and is required for the phosphorylation of synaptosomal proteins. Therefore, the objective was to compare levels of brain calcium and calmodulin in the Japanese quail. Brain extracts from embryonic days 11, 15, hatch, and 5 days post-hatch (n = 7/group) were analysed for calcium, protein and calmodulin. Despite increases in protein between embryonic (X = 0.126 and 0.145 mg/mg wet wt) and hatched groups (X = 0.183 and 0.221 mg), no significant increases in calmodulin were observed (237-279 ng/mg protein). Calcium levels in the brain were U-shaped with low levels at embryonic day 1 (341 micrograms/mg wet wt) and post-hatch day 5 (315 micrograms/mg wet wt) with higher levels on embryonic day 15 (425 micrograms/mg wet wt) and at hatch (433 micrograms/mg wet wt). Calmodulin levels do not show a developmental pattern similar to calcium and protein levels or with reports of brain activity.

Intrinsic differences in the transplantability and outgrowth potential of DMBA-induced rat mammary tumors.

Only a small proportion of DMBA-induced rat mammary tumors are transplantable in cleared (gland-free) mammary fat pads. To enhance their transplantability, several host factors were modified: age, parity, hormone levels, and tissue integrity of the fat pad. Our 2 important findings were that (1) there were individual differences between the tumors in their transplantability and potential to proliferate normal and hyperplastic outgrowths, and (2) these differences were determined by their inherent potential and not by their environment. These intrinsic differences among the tumors are consistent with the concept that properties of a given type of tumor are independently variable.

Production of monoclonal antibodies against avian LHRH-I.

Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 micrograms of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 micrograms of the immunogen in 75-cm2 tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse.

Quantitation of calmodulin in the mammary gland of the lactating sow.

It has been suggested that calmodulin, a calcium-binding protein, has a functional role during milk secretion. High levels of calmodulin are present during lactation in rat mammary glands and a substantial increase has been observed in the bovine mammary gland prior to parturition. In the sow, regressed glands involute while suckled glands remain highly active even though they are under the same hormonal influence. In this study, tissue samples were taken from suckled and regressed glands of the same sow at both peak and late lactation. Calmodulin and total protein were measured in tissue homogenate supernatants. Residual milk was apparent in regressed glands during mid lactation but not in the same glands by late lactation. Calmodulin levels in tissue were the same for both suckled and regressed glands. There was a slight but non-significant increase in the tissue calmodulin level from peak to late lactation. Protein levels declined significantly from mid to the late stage of lactation. There was no change in protein level between the suckled and regressed glands. Calmodulin may be responsible for casein phosphorylation and/or the mediation of prolactin action on the gland. The precise regulatory mechanisms relating hormonal control to calmodulin levels during lactation need further investigation.

Evidence of calmodulin in bovine milk with high somatic cell counts.

The objective was to determine whether calmodulin was present in bovine milk with high SCC. A highly specific antibody against calmodulin was developed in rabbits and affinity purified. Enzyme-linked immunoassays were used to determine the presence of calmodulin in the whey and casein fractions in milks with SCC ranging from less than 50,000 to greater than 1,000,000. Percentages of total protein and casein were determined. Calmodulin was detected in some casein fractions regardless of level of SCC. In the whey fraction, calmodulin was positively correlated with increased SCC. Calmodulin may be released into the milk as a result of the elevated proteolytic activity, which is evident in mastitis or as a result of leakage from the serum. This provides further information on the cellular and biochemical changes that occur in the diseased udder. Total protein increased with the increase in SCC while there was an inverse relationship of SCC to the percent casein.

Factors influencing differential growth of rat mammary tumor fragments and cells transplanted in gland-free and gland-containing mammary fat pads.

Transplants from eight primary tumors induced by 7,12-dimethylbenz(a)anthracene were placed into gland-free and gland-containing inguinal mammary fat pads of syngeneic female hosts. Samples were transplanted as 1 mm3 fragments or as cell suspensions of enzymatically dissociated tumors averaging 1.5 X 10(5) cells/ml. Similar differential growth patterns resulted from both types of transplants. In samples from two of eight tumors fragments produced more tumors than did dissociated transplants, regardless of fat pad condition. In one case dissociated transplants gave rise to more tumors than did fragments. Samples from two of eight tumors produced no differences in tumor growth from fragments and dissociated transplants. Samples from the remaining two showed low transplantability. Overall, no significant differences in tumor growth could be attributed to transplant treatment or fat pad condition. Gland-containing fat pads inhibited ductal outgrowth following transplantation while gland-free fat pads favored it. From the data, the greater the lability of the primary tumor, the greater the influence of the host microenvironment on the outgrowth potential of the tumor.