One hundred consecutive insulin-treated diabetic patients with a normal serum creatinine value admitted to a Diabetes Day Care Unit were investigated. Because we were studying microalbuminuria, 20 patients with persistently positive Albustix tests had to be excluded. The albumin/creatinine clearance ratio, a test used to determine albuminuria in renal diseases since 1973, determines the loss of albumin from the total glomerular surface and has, in contrast to other methods for measurement of microalbuminuria, the advantage of being uninfluenced by diuresis. The albumin/creatinine clearance ratio, in comparison with random urinary albumin concentration, correlated well with 24-h urinary albumin excretion (r=0.74, p<0.001). Furthermore, the albumin/creatinine clearance ratio correlated (p<0.001) with glycemic control (HbA1c; r=0.41) and with blood pressure (diastolic BP; r=0.50). The ratio (×10-3) was higher in patients with HbA1c≥8.7% (median value) (0.02±0.01, p<0.01), or diastolic BP≥90mmHg (0.05±0.02, p<0.001), or both (0.07±0.01, p<0.001), compared with those with HbA1c<8.7% and diastolic BP<90mmHg (0.003±0.001). The albumin/creatinine clearance ratio, determined from a single blood and urine sample, is a convenient and experienced test useful when screening for microalbuminuria. The ratio is related to glycemic control and blood pressure.
A sensitive high-performance liquid chromatography (HPLC) method was used for the determination of thiamin and its phosphate esters (thiamin triphosphate, TTP; thiamin diphosphate, TDP; and thiamin monophosphate, TMP) in normal human whole blood. The blood specimens deproteinized with cold ca. 7% trichloroacetic acid, were applied to a reversed-phase HPLC column (Shim-pack CLC-ODS) equipped with a spectrofluorometric system. Picogram levels of free thiamin and its phosphate esters were determined after conversion to the corresponding thiochrome derivatives in a post-column reaction with alkaline ferricyanide. The contents of thiamin and its phosphate esters were 24.8 (±4.2), 55.5 (±7.6), 1.6 (±0.8) ng/ml (N=8), and a trace amount for TTP, TDP, TMP, and thiamin, respectively. The most suitable method of sample preparation for HPLC determination of blood thiamin and its phosphate esters was found to be immediate deproteinization of the blood with trichloroacetic acid, followed by centrifugation and storage of the supernatant at -30°C until analysis could be performed. Although the total thiamin contents in human blood determined in the present study were similar to those reported previously using different procedures, the percentage of thiamin triphosphate (≅30%) was found to be high, which might be due to the existence of the enzyme which catalyses the synthesis of TTP from protein-bound TDP and ATP in the presence of Mg2+ and serum cofactor in human whole blood.
In order to investigate the pathogenesis of vitamin K deficiency in early infancy, we measured urinary γ-carboxyglutamic acid (Gla) by high performance liquid chromatography, plasma and urinary bone Gla-containing protein (BGP) by radioimmunoassay, and vitamin K-dependent coagulation activity by Normotest, in newborn and 1-3-month-old infants. The infants were divided into two groups, one of which received vitamin K orally at birth and at the 7th day thereafter, and the other, which did not receive vitamin K, was taken as the control. Urinary Gla level in the control group was fairly steady during early infancy, whereas in the vitamin K-administered group urinary Gla increased with age and was significantly higher than that of the control group from the neonatal period to 1 month of age. The difference in urinary Gla level between the two groups was maximum at 1 month of age. Coagulation activity in both groups increased with age and the activity in the vitamin K-administered group was slightly higher than that in the control, however, the slopes for age versus coagulation activity between the two groups were not significantly different. Plasma BGP level in the control group increased linearly during the neonatal period and then significantly decreased at 1 month of age. The urinary BGP level in the control group increased linearly from the neonatal period to 3 months of age. On the other hand, the plasma BGP level in the vitamin K-administered group was significantly lower compared with that in the control group from 2 days of age to 1 month, and the urinary BGP level was also lower than in the control during the neonatal period but higher than in the control at 1 month of age. Urinary Gla mostly increased at 1 month of age when vitamin K was administered, indicating that there are newly synthesized non-γ-carboxylated, vitamin K-dependent proteins which are changed to the γ-carboxylated form and excreted in urine as free Gla by vitamin K administration, especially at 1 month. The decreased BGP level at 1 month of age in the control group suggests that BGP in plasma deposits to bone, because there is no significant change detected in urinary BGP and Gla. Moreover, the high urinary BGP level at 1 month of age in the vitamin K-administered group suggests that excessive BGP, which is formed as the result of increased bone turnover at 1 month, is the source of this high urinary level. Therefore it is speculated that increased BGP turnover to regulate mineralization at 1 month of age, and induce acceleration of vitamin K requirement of the body and consequently infants easily manifest vitamin K deficiency if there are risk factors decreasing vitamin K intake.
Serum cholesterol (TC) and triglycerides (TG) levels were measured in residents (aged 20-69 years), of Kumamoto Prefecture, Japan, at health screenings in 1983-1985. Results were compared with those obtained in the same area in 1973-1976. TC showed a significant increase of 6-12mg/dl in 35-39 and 45-49-year-old males, and in 25-44-year-old females. TG showed a significant decrease of 9-25mg/dl in 35-44-year-old males, and in 45-69-year-old females. A nutritional study was conducted, which showed that intake of protein, fat, and animal fat had increased. This result agrees with the annual changes reported in daily per capita nutritional intake of the Japanese. Although the life-style and particularly the diet of the Japanese have been changing rapidly, there has been relatively little change in TC levels. This fact may help to explain why the mortality rate for cardiac disease in Japan is still one of the lowest among the developed nations.
Lipid peroxide levels in various parts of normal skin, both exposed and unexposed, and the skin lesions of twenty-eight patients with various inflammatory and non-inflammatory diseases were assessed by our modified TBA method. A linear correlation between the concentration of skin homogenate and lipid peroxide level was obtained for normal skin. Exposed normal skin showed a higher lipid peroxide level than unexposed skin. Lipid peroxide levels in the skin lesions from patients with inflammatory skin diseases such as acute eczema, hair dye contact dermatitis, radiation-dermatitis, and traumatic open wound in active stages were markedly greater than those in the corresponding areas of the normal controls. However, inflammatory skin lesions such as pruritus and vitiligo did not show any significant change. The present study suggests that, since skin is always in contact with oxygen and usually exposed to ultraviolet light, not only in thermally-generated lesions but also in skin inflammatory sites in their active stages, membrane damage is readily induced, leading to increased levels of lipid peroxides.
Effects of pyridoxal 5′-phosphate on enzymatic reactions of cross-link formation in collagen and elastin were studied. In this study, we observed that pyridoxal 5′-phosphate combined with β-aminopropionitrile to competitively reverse the inhibition of activities of vitamin B6-dependent enzymes brought about by β-aminopropionitrile in vitro. In addition, we observed that a sufficient dose of pyridoxine hydrochloride prevented the occurrence of dissecting aneurysm of SD rats fed β-aminopropionitrile. From the above results, we assume that β-aminopropionitrile inhibited the lysyl oxidase by binding with the pyridoxal 5′-phosphate that is necessary for the activation of lysyl oxidase.
The biochemical changes in male Wistar rats with galactosamine-induced (total dose —2.78mmol/kg body mass, divided equally into three i.p. injections) hepatitis maintained on high-protein (46% casein) or glucose (63%) diets were studied. The diets containing glucose instead of starch reduced the effects of galactosamine on serum aspartate and alanine aminotransferase activities and on 5′-nucleotidase, K+, Na+- and Mg++-adenosine triphosphatase activities in liver plasma membrane preparations. A diminished content of reduced glutathione in liver homogenates of the galactosamine treated animals fed glucose-containing diets was established. Liver microsomal cytochrome P-450 concentration showed no significant changes. The activities of some intestinal disaccharidases were increased, however, by galactosamine treatment. The data suggest that intestinal epithelium may also be a target in this hepatic lesion.
Time related changes in distributions of body fluid volumes were studied in rats with either of two types of Goldblatt hypertension; two-kidney, one-clip (2K1C) and one-kidney, one-clip (1K1C) models. Plasma volume (PV), extracellular fluid volume (ECF), and total body water (TBW) were measured on days 1, 7, and 28 after renal artery stenosis. Mean arterial pressure (MAP) and plasma renin activity (PRA) were also determined in conscious rats. In the 2K1C rats each fluid compartment on any experimental day did not differ from the sham controls except for a decrease in PV of the clipped rats on day 1. MAP in the 2K1C increased significantly (p<0.05) only on day 28 as compared with the controls. In the 1K1C rats the tendency toward an increase in PV and ECF on early days was followed by a significant (p<0.05) elevation in ECF on day 28. MAP was higher in the clipped rats than in the controls on days 7 and 28. There were no differences in PRA between the clipped rats and the controls either in 2K1C or 1K1C throughout the study. These results suggest that alterations of body fluid volumes might be involved in the development and maintenance of hypertension in the 1K1C rats, but not in the 2K1C ones. In contrast, the role of the renin-angiotensin system was rather negligible in these two models, as judged from the PRA levels.
We studied the effect of hypoxia on lipid accumulation in cultured fibroblasts from normal rabbits (normal fibroblasts) and in low density lipoprotein (LDL) receptor-negative fibroblasts from WHHL (Watanabe heritable hyperlipidemic) rabbits (WHHL fibroblasts). These cells were incubated in medium with normolipemic rabbit serum (NRS) or hyperlipemic rabbit serum (HRS). The cells were incubated in a humidified atmosphere of either 20% O2, 75% N2, and 5% CO2 (control cells) or 2% O2, 93% N2, and 5% CO2 (hypoxic cells). After 48h incubation of normal fibroblasts in medium with 20% NRS, free fatty acid (FFA) levels were increased slightly and the triglyceride (TG) level markedly in hypoxic cells. In the medium with 20% HRS, in addition to the increased FFA and TG levels, the free cholesterol level was increased slightly and the esterified cholesterol level markedly in hypoxic cells. Moreover, in WHHL fibroblasts, which lack LDL receptors, cellular lipid accumulation was also observed after 48h incubation in the medium with 20% HRS, and hypoxic incubation enhanced the cellular cholesterol and TG accumulation, as in normal fibroblasts. These results suggest that under hyperlipemic conditions, non LDL receptor-mediated uptake of lipoproteins plays a major role in cellular lipid deposition and that tissue hypoxia promotes lipid accumulation in peripheral cells by the LDL receptor-independent mechanisms.