In this study, we report 64Cu PET/CT results on biodistribution, dosimetry, and hepatic removal kinetics following both intravenous and oral administration of the radiotracer in healthy humans.
64 Cu Biodistribution
Following absorption in the intestines, copper is transported into the portal blood circulation by the ATP7A transporter located in the basolateral membrane of the enterocytes . In the portal vein, copper is bound to albumin, in particular, and to other plasma proteins in a highly exchangeable pool [18, 19]. Albumin-bound copper in systemic plasma has a half-life of 10–20 min  and is effectively extracted during the hepatic first pass (> 80%) . In the present study, high uptake of 64Cu from the systemic circulation after intravenous administration was also observed in other tissues characterised by high expression of the CTR1 transporter such as the pancreas, intestinal walls, and kidneys . After intravenous administration, 64Cu was not excreted in urine and only a negligible amount was detected in faeces during the 20-hour observation period.
After oral administration, the biodistribution was dominated by efficient hepatic first pass extraction of 64Cu , whereas uptake in organs other than the liver, kidneys, and red bone marrow was negligible when compared with intravenous administration. Moreover, the total %AD taken up from the intestines and measured in source organs did not exceed 50%, which is in accordance with net intestinal copper absorption studies in pigs and humans [19, 23]. It should be noted that the intestinal absorption of copper is affected by the dietary composition and our results therefore only reflect conditions in the 6 h fasting state .
In the liver, copper is incorporated in ceruloplasmin (biological half-life: 13 hours) and then redistributed into the systemic circulation 2–3 days after administration, creating a second peak in blood concentration, also known as the ceruloplasmin wave [25, 26]. In the present study, the blood concentration of 64Cu steadily increased after the peak following administration, reflecting copper incorporation into ceruloplasmin. The arterial blood to plasma radioactivity ratio was approximately 55% and increased over the first 90 min, possibly reflecting copper uptake in erythrocytes by the anion exchanger located in the erythrocyte membrane .
64 Cu Dosimetry
Dosimetry estimates for intravenous administration showed that the liver was the most critical organ, followed by the small intestines. Reports of dosimetry estimates for intravenous administration of 64Cu differ to some extent. In the present study, radiation exposure to the liver and intestines was considerably higher and the effective dose twice of what was previously reported in patients with prostate cancer [5, 6]. This difference is likely caused by different analytical approaches rather than altered biodistribution of 64Cu in patients with prostate cancer compared with healthy subjects. However, our results, based on the newest phantoms and tissue weighing factors [14–16], are in agreement with the observations made by Avila-Rodriguez et al. in healthy participants .
To our knowledge, dosimetry estimates for oral administration of 64Cu have not previously been reported. As expected, the radiation dosimetry of 64Cu after oral and intravenous administration differed significantly. While the liver was exposed to a high radiation dose after oral administration, equal or higher doses were received by the intestines due to high amounts of unabsorbed radiotracer. In this context, it is important to acknowledge that the radiation dose to the intestines depends on the individual intestinal transit time, unlike for intravenous administration; in our study, one participant received 925 µGy/MBq to the right large intestine. Consequently, radiation dosimetry of 64Cu by oral administration may differ substantially between individuals necessitating a cautious approach to total oral dose used in future studies. Based on our results, the total radiation dose received by the reference gender-averaged adult after an oral ingestion of 50 MBq 64Cu amounts to 5.6 mSv, ensuring less than 50 mSv absorbed by a single organ. This dose is sufficient to obtain high-quality PET images, and may still be reduced by at least 50% with new digital PET systems yielding faster time-of-flight timing resolution and higher NEMA sensitivity. Thus, 64Cu PET using intravenous or oral administration is suitable for studying copper metabolism in humans.
The use of radioactive Cu isotopes to assess copper metabolism in humans was introduced decades ago and many studies on this topic have been published since then [28–31]. Most recently, Czlonkowska et al. showed that measurements of 64Cu in blood and in urine following intravenous injection accurately distinguished between patients with Wilson’s disease and heterozygote controls . The obvious advantage of 64Cu-copper PET/CT is however, the potential for assessing also the hepatic uptake, accumulation, and turnover; this includes oral administration where the biodistribution is dominated by first pass extraction by the liver, as demonstrated in the present study. In addition, the PET/CT data on the accumulation of protein-bound 64Cu reported in this study provides valuable knowledge to help interpret unwanted copper loss in relation to the increasing research on 64Cu radiopharmaceuticals.
Peng et al. assessed hepatic copper kinetics in rats using 64Cu PET/CT , revealing some noteworthy differences between rodents and our human participants. For example, cardiac uptake of 64Cu was substantial in rodents whereas it was negligible in our human participants. Results from rodent copper studies can therefore not be easily translated to human conditions. In the present study, we were able to quantify the hepatic removal kinetics of 64Cu using dynamic PET/CT with arterial blood. The hepatobiliary excretion of 64Cu is slower than e.g. bile acids  but importantly, the properties of the 64Cu isotope allows for long-term studies of copper metabolism in humans.