Serum urate level is strictly regulated by renal urate reabsorption and secretion through the urate transporters at the apical or basement membrane of renal tubular cells. Since urate transporters could be a therapeutic target for hyperuricemia, it is required to observe the transporting dynamics in living cells and the rapid evaluation is needed to screen novel inhibitors. With the aim of development a high-throughput biosensing method for analysis of urate transport, a fusion protein probe was constructed in this study. The fusion construct was consisted of uricase and hydrogen peroxide-dependant fluorescent protein （HyPer）. Uricase produces hydrogen peroxide when oxidizes urate, and the uricase-HyPer fusion protein could detect urate specifically with fluorescence.
In this study, fusion protein-coding plasmid DNA was electroporated to monkey renal epithelial-like cell line COS7 that stably expressed urate transporter 1 （URAT1）. The transfected COS7 cells were added into buffer containing 0-200 μM urate or adenine and its time-dependant fluorescence unit was determined. The uricase-HyPer/COS7-URAT1 cell showed the significant increase of fluorescence when 10-200 μM of urate was applied, except for adenine. The uricase-HyPer fluorescence was suppressed with 0.5-10 μM supplement of URAT1 inhibitor, benzbromarone. These results suggested that urate uptake by transporter was occurred and uricase-HyPer could detect the urate uptake specifically. In conclusion, fluorometric live-analyzing method for characterization of urate transport was developed. The present method is applicable to screening for novel urate transporter inhibitor, regulating serum urate level.

View full abstract

　Hyperuricemia is an independent risk factor for cardiovascular disease in hypertensive patients; therefore, management of serum uric acid (SUA) levels is important. We investigated the status of SUA levels in hypertensive patients. Subjects were 667 outpatients (359 women; mean age, 66.4±12.6 years) undergoing treatment for hypertension. On average, they took 2.3 antihypertensive drugs. Calcium-channel blockers (82.8%) and angiotensin-II receptor blockers (75.1%) were the predominantly prescribed agents, and thiazide and loop diuretics were given to 22.0% of patients. The mean SUA level was 5.6 mg/dL. Hyperuricemia was defined as SUA > 7mg/dL or the use of uric acid-lowering drugs, and was observed in 23.4% of subjects. Achievement of SUA ≤ 6mg/dL was observed in 63.1% of subjects. In total, 52.5% of patients taking ≥3 antihypertensive drugs took diuretics, and the prevalence of hyperuricemia in this group was 37.3%. Although 24.5% of patients taking diuretics also took uric acid-lowering drugs, achievement of SUA ≤ 6mg/dL was possible in only 51.0% of individuals. These results suggest that the control of SUA levels in hypertensive patients is insufficient, especially in those taking multiple antihypertensive drugs (including diuretics). Encouragement of lifestyle modification, the use an the appropriate class of antihypertensive drug, and pharmacological intervention to lower SUA levels are required to reduce the risk of cardiovascular disease in hypertensive patients.

View full abstract

The frequency of hyperuricemia in the Japanese male population has been estimated by using data of regular health checkups conducted by companies or thorough medical examinations. These data have shown that the frequency of hyperuricemia is lower in men in their 40s or older than in men in their 30s. In general, the rate of obesity, which closely correlates with hyperuricemia, is higher in men in their 40s or older than in men in their 30s in Japan. Furthermore, alcohol consumption, which also closely correlates with hyperuricemia, is also higher in men in their 40s or older. To elucidate the cause for the lower frequency of hyperuricemia in older men, we used data of health insurance claims to obtain the rates of prescribing urate-lowering drugs in different age groups. The rates of prescribing urate-lowering drugs increased as age increased (30s : 1.2%, 40s : 3.3%, 50s : 5.8%, 60s : 9.8%), and these rates seem to provide an explanation for the difference in frequency of hyperuricemia among age groups. Our results suggest that the lower frequency of hyperuricemia in men in their 40s or older than in men in their 30s is largely due to treatment with urate-lowering drugs.

View full abstract

Here, we describe quantitative analysis of plasma uric acid (UA) levels using an improved HPLC-UV method, which provides highly sensitive analysis using microliter volumes of plasma. Phosphoric acid aqueous solution (pH 1.8) was used as the mobile phase. The quantitative analysis of oxypurines in plasma using a polymeric-ODS with high-carbon content (ca.17%) column (Φ4.6×250mm) was accurate and reproducible. The reproducibility of the UA analysis had a determination limit of 0.14mg/dL and a coefficient of closing variation of 0.98%.
The results by our method confirmed that the plasma UA levels were increased in mice with oxonic acid-induced hyperuricemia compared to normal mice, and that allopurinol reduced the plasma UA levels in these mice. This method allows the measurement of plasma uric acid levels using small plasma samples.

View full abstract